Team:CU-Boulder/Notebook/CC9 Team/Induced

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Revision as of 23:53, 13 October 2014

Week 1 5/12/14

Made overnight cultures of….

- Qi gRNA P44251 (AmpR) NOTE: plate from Andrew is labeled P44231 - Qi wr cas9 P44250 (AmpR) - BW 23115 Kan casA (KanR)

5/13/14 Glycerol stocks - Stocks made from overnight cultures (3 from each) - 500 μL bacteria + 500 μL 40% glycerol stock

Glass tube containing #P44250 broke and was discarded – overnight repeated

Mini Prep Protocol done per manufacturer’s instructions with following exceptions: -Done with P44250 on 5/13/14 -Done with P44251 on 5/14/14 -Step 1: Cultures split into 3 separate 1.5 mL aliquots. Supernatant was removed from all three, pellets resuspended in a small amount of EB Buffer and moved into single tube. Centrifuged again and supernatant was removed before proceeding to Step 2.

5/14/14 _t Nano Drop of Mini Prep Sample 260/280 Ratio 260/230 Ratio DNA Concentration P44250 1.94 2.17 49.4 ng/μL P44251 1.94 2.47 52.7 ng/μL _t

Samples were blanked with EB Buffer

Restriction Digest - NEBcutter V2.0 used to check digestion cut sites (done in silico) - Digest reaction performed as shown below (from NEB) - Incubated for 1 hour at 37°C - No heat inactivation performed

_t P44250 P44251 Cutsmart Buffer 5.0 μL 5.0 μL XbaI 1.0 μL 1.0 μL XhoI 1.0 μL -- SpeI 1.0 μL -- EcoRI-HF -- 1.0 μL DNA (200 ng) 4.0 μL 4.0 μL MQ H2O 38.0 μL 39.0 μL Total Volume 50.0 μL 50.0 μL _t

Agarose Gel Electrophoresis -1% agarose gel prepared -2 μL loading dye added to 18 μL of each sample for loading into each lane -1Kb+ DNA Ladder used -Overnight staining in EtBr done to better visualize faint bands

Lane 1: 2-Log DNA Ladder Lane 2: P44250 digest Lane 3: P44251 digest

P50 plasmid should have 3 segments after digestion: 4221 bp, 1740 bp, 970 bp P51 plasmid should have 2 segments after digestion: 540 bp, 2045 bp -This is demonstrated in the gel image shown below.

Plate Streaking -Used to check the TetR gene in the P44250 plasmid -2 mL LB containing 50 mg/ μL Tet added to plate and allowed to soak in -Plates left at room temperature overnight

- LB only plate – negative control - Amp plate - Amp + Tet - Tet

5/15/14 Dry Lab - gRNA designed for Kanamycin - Plasmid design – considering Gibson assembly and cPEC

Analysis of streak plates from 5/14/15 Results: (See Below) LB: Slightly less growth than Amp plate Amp plate: Normal Growth Amp + Tet: No bacteria present Tet: No bacteria present Results were inconsistent with expectations. There is some concern regarding the potency of the tetracycline used. Further research revealed that the TetR gene is a repressor for TetO which part of the bi-directional promoter for cas9. Thus TetR is inhibited with presence of tetracycline. We will use TetR as a regulator for cas9.

5/16/14 Dry Lab - gRNA designs modified - Primer design for Gibson assembly of our plasmid

Week 2 5/19/14 Transformation: 1 μL P44250 (cas9) plasmid added to K12 E. coli strain Heat shock done according to protocol (45 sec @ 42°C) Cultures incubated at 37°C for 1.5 hours 4 Plates with K12 and the plasmid were plated by streaking as follows: - Amp, Amp (½ plate with 1:10 dilution, ½ plate 1:1), Amp + Kan, Amp + Kan (½ plate with 1:10 dilution, ½ plate 1:1) 5/20/14 Plates from 5/19/14 (shown below).

Two individual colonies were taken and grown overnight from the Amp plate (marked with “x” in upper left plate of above image). There was no growth on the two plates with Amp and Kan (K12 strain does not contain Kan resistance).

5/22/14 Transformation: (Chemically competent BWF+ cells with P44250 plasmid) 1 μL P44250 (cas9) plasmid added to conjugated BWF+ strain Transformation protocol followed with following exceptions: -200 μL SOC added to each sample -Incubated for 2 hours -1:10 dilution made (10 μL into 90 μL SOC) -Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution

5/23/14 Arrival of primers previously ordered All were reconstituted in MQ H2O to 100 μM concentration

Results of plates from 5/22/14: - BFW+ w/ P50 plasmid: growth at full and 1:10 dilution (Top left of image) - BFW+ w/ P50 plasmid full strength: No growth (Bottom left of image) - BW only: full strength showed minor growth (Bottom right of image) - BW only: No growth at full and 1:10 dilution (Top right of image) (*This was an unexpected result – it may be that these two plates were mislabeled)

Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 μL liquid culture overnight with the following antibiotics: - AMP - Kan - AMP + Kan - Tet

5/24/14 As shown the image below, overnight cultures from 5/23/14 are all cloudy demonstrating bacterial growth.

PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid *DNA samples diluted so each contains 0.5 ng/μL

_t Cas9 CMR Q5 Master Mix volume 25 μL 25 μL dcas9 Primer volume 2.5 μL -- dCMR Primer volume -- 2.5 μL DNA added 2.5 μL 2.5 μL Q5 Polymerase volume 0.5 μL 0.5 μL Nuclease Free H2O 20 μL 20 μL Final Reaction Volume 50.0 μL 50.0 μL _t PCR Settings: 30 cycles as follows - 98 °C Denature 30 sec - 70 °C Anneal 30 sec - 72 °C Extension 2.5 min

Week 3

5/25/14 Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 μL EtBr. Lane 1: 5 μL 2-Log DNA Ladder Lane 2: CMR sample* (~2 Kb size) Lane 3: Cas9 sample* (~5 Kb size)

Each contained 8 μL reaction sample + 2 μL loading dye

Gel Results:

Lane 1 – Ladder Lane 2 – Although band of correct size visible for CMR sample (~2Kb), there may be contamination Lane 3 – No bands visible – This is perhaps due to having the extension time too short in the PCR reaction (Advisors suggest 60 sec per 1000 bases).

5/27/14 Repeat of PCR using same protocol as listed on 5/24/14 except for different times on the thermocycler as follows: _t Cas9 CMR Initial Denature 98°C/2 min 98°C/2 min Denature 98°C/30 sec 98°C/30 sec Anneal 69°C/30 sec 72°C/30 sec Extend 69°C/ 2.5 min 72°C/2.5 min Final Extension 72°C/6 min 72°C/5 min _t

Thermocycler containing CMR stopped after 1 cycle and held at 98°C for ~3hours. Machine was reset and run. This sample may no longer be viable.

5/28/14 Dry lab – primer designs Repeat of PCR for the CMR reaction, same as described on 5/24/14 and shown below. _t Reactants CMR Q5 Master Mix 25 μL dCMR Primer 2.5 μL Q5 Polymerase 0.5 μL CMR DNA 2.0 μL Nuclease Free H2O 20 μL Final Reaction Volume 50.0 μL _t 5/29/14 Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.

_t Lane Sample Amount Loading dye amount 1 2-Log DNA Ladder 0.5 μL -- 2 Cas9 5.0 μL 1.0 μL 3 CMR 1 (from 5/24) 5.0 μL 1.0 μL 4 CMR 2 (from 5/27) 5.0 μL 1.0 μL 5 CMR 3 (from 5/28) 5.0 μL 1.0 μL _t Results from Gel (shown below): Lane 2: Cas9 band visible of appropriate size (approximately 5,000 bp) Lane 3: CMR 1- slight smearing Lane 4: CMR 2 – smearing present Lane 5: CMR 3- clean, no smearing All CMR lanes have band present of correct size (approx. 2,000 bp)

Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following the protocol. Following amounts: - Cas9: 110 μL buffer with 0.1103 g sample - CMR1: 53 μL buffer with 0.0533 g sample - CMR2: 90 μL buffer with 0.0904 g sample

Nanodrops performed to determine DNA concentration and purity with the following results:

_t Sample ng/ μL 260/280 260/230 **ng/ μL **260/280 **260/230 Gel-Cas9 7.3 2.20 0.57 5.2 2.06 0.52 Cas9 483.2 1.78 0.72 36.3 1.85 1.72 Gel-CMR1 9.6 2.02 0.63 6.5 2.35 0.90 Gel-CMR2 8.3 2.06 0.83 5.0 2.21 0.69 CMR2 618.8 1.66 0.61 44.5 1.89 1.41 CMR 3 535.8 1.73 0.61 17.8 1.89 1.31 CMR1 516.6 1.69 0.61 68.5 1.84 1.74 _t

Trial 2 completed on 5/30/14 shown in these columns

Values for the ratios should be between 2.0 and 2.2 for a pure sample. The values for Trial #1 are much too low. In order to increase sample purity, a DNA cleanup is performed following the protocol given in the E.Z.N.A. gel extraction kit in amounts shown below. - 25 μL samples - 25 μL binding buffer - Washed 2x with 700 μL SPW - 30 μL elution buffer added, allowed to rest 4 minutes - Centrifuged on max for 2 min

5/30/14 After the DNA cleanup protocol – nanodrop performed again with results listed in above table as Trial #2

Restriction Digestion performed on Cas9 and CMR1 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts: 26 μL sample 1.0 μL DPN1 (100 fold dilution) 3.0 μL 10X CutSmart Buffer

Gibson Assembly performed to join pSB1C3 backbone with cas9 PCR product. Protocol for Gibson was followed for the reaction. _t Reactants Volume Gibson Master Mix 10 μL Cas9 1.6 μL (50 ng) Cmr 2.4 μL (143 ng) Nuclease Free H2O 6 μL Final Reaction Volume 20.0 μL _t

Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.

Week 4

6/2/14

Grow Gibson colonies in 6 mL LB media + 30 μL chloroamp

Transform Gibson colonies following protocol and with amounts as shown: - BWF+ w/cas9: 15 μL into 135 μL SOC - BW w/cas9: 15 μL into 135 μL SOC - BWF+ (no cas9): 30 μL into 270 μL SOC - BW (no cas9): 30 μL into 270 μL SOC

90 μL of the above four reactions plated as follows and incubated: - BWF+ w/cas9: Amp + Kan - BW w/cas9: Amp + Kan - BWF+ (no cas9): (2 Plates) Amp + Kan and Kan only - BW (no cas9): (2 Plates) Amp + Kan and Kan only - All four reactions also plated together on Quad plate with Chloramp - BWF+ (Not transformed) on Kan + Tet (plated both at full strength and as 1:10 dilution)

6/3/14

Analysis of Plates from 6/2/14: Only one colony was present on Gibson plate (BWF+ w/cas9) This colony was taken and suspended in O/N culture (LB and Choroamp) MiniPrep performed on the O/N cultures *Renamed the product GAcasC (Gibson Assembly w/cas and Chloroamp) NanoPrep on GAcasC

_t ng/ μL 260/280 260/230 201.0 1.88 2.24 _t Digest performed according to protocol on GAcasC and P50 plasmid using EcoRV and BamHI as follows:

_t Reactants GAcasC P50 Buffer 3 2.5 μL 2.5 μL EcoRV 0.5 μL 0.5 μL BamHI 0.5 μL 0.5 μL BSA (1:4 dilution) 1.0 μL 1.0 μL DNA (250 ng) 1.25 μL 5.0 μL MQ H2O 19.25 μL 15.5 μL Total Volume 25.0 μL 25.0 μL _t

1% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 120V for ~1 hour.

_t Well Loaded 1 DNA Ladder 2 GAcasC digest 3 CmR1 Dpn1 product 4 Cas9 Dpn1 product 5 P50 digestion product _t Gel Results: Lane 2 should have multiple products which are not present in the gel. The other lanes are as expected.

PCR using Q5 is used to amplify the P51 CRISPR backbone using primers CRE1 & CRE4 provided by Andrew. Primers were diluted 1:10 from 100 mM stock

_t Reactants Volume Q5 Master Mix 25 μL CRE1 Primer 2.5 μL CRE4 Primer 2.5 μL P51 DNA 2.0 μL Q5 Polymerase 0.5 μL Nuclease Free H2O 17.5 μL Final Reaction Volume 50.0 μL _t

Thermocycler set as follows for the reaction:

_t Initial Denature 98°C/30 sec Denature 98°C/20 sec Anneal 60°C/20 sec Extend 72°C/3 min _t

Restriction Digestion performed on P51 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts: 26 μL sample 1.0 μL DPN1 (100 fold dilution) 3.0 μL 10X CutSmart Buffer

PCR performed to dimerize each of the gRNA primers previously designed (729, 666, 756, SC1, and SC2) following the cPEC protocol and using the following amounts:

_t Reactants Volume Q5 Master Mix 25.0 μL Primer 1.7 μL Primer 2.0 μL Q5 Polymerase 0. 5 μL Nuclease Free H2O 19.5 μL Final Reaction Volume 50.0 μL _t

Nanodrop performed on some of these products (due to timing) to check for purity and concentration (results below). It is assumed that the other samples have high enough DNA levels to proceed.

_t Sample ng/ μL 260/280 260/230 729 405.5 1.78 0.67 SC2 393.2 1.80 0.67 CasC 455.2 1.5 0.60 _t

cPEC performed to combine the P51 amplification and the each of the dimerized gRNAs separately following the cPEC protocol and using the following amounts:

_t Reactants Volume Q5 Master Mix 12.5 μL Insert (our primer dimers) 1.7 μL Template DNA 2.0 μL Q5 Polymerase 0.25 μL Nuclease Free H2O 10.05 μL Final Reaction Volume 25.0 μL _t

Repeat of Gibson to combine cas9 and CmR (pSB1C3 backbone) with cas9 PCR product. Gibson protocol was followed for the reaction and included a positive control (same as reaction completed on 5/30/14).

_t Reactants Volume Gibson Master Mix 10 μL Cas9 1.6 μL (50 ng) Cmr 2.4 μL (143 ng) Nuclease Free H2O 6 μL Final Reaction Volume 20.0 μL _t

Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.

O/N cell cultures made from plates in order to perform a Tet titration tomorrow. 2 tubes of each were made by taking a single colony and mixing with 5 mL of LB and antibiotics as listed below before incubation.

_t Sample Antibiotic BWF+ w/ cas 5 μL Kan + 10 μL Amp BW w/ cas 5 μL Kan + 10 μL Amp BWF+ 5 μL Kan BW 5 μL Kan BWF+ (not transformed) 5 μL Kan + 10 μL Tet _t

6/4/14

Tet titration on O/N cultures was performed by using different concentrations (from 0-1000 ng/mL) of anhydrotetracycline plus a chloramp control.

Cancelled as cell growth had already neared plateau values

_t Sample 0 ng/mL 0.5 ng/mL 5 ng/mL 20 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL Chloro BW 2.38 2.401 2.389 2.382 2.386 2.398 2.373 2.379 BWF+ 2.379 2.376 2.428 2.390 2.378 2.365 2.378 2.386 BWcas 2.391 2.356 2.367 2.355 2.366 2.330 2.362 2.382 BWF+ cas 2.438 2.447 2.448 2.434 2.436 2.415 2.419 2.415 BWF+ Tet 2.135 2.146 2.141 2.152 2.128 2.127 2.134 2.134 _t

New O/N cell cultures made to do the titration on 6/5/14. Same amounts as shown above were used except only one of each was made. No cells added to O/N culture on 6/4/14, that is why procedure was repeated.

6/5/14

Since the Gibson Assembly does not seem to be working, cPEC was performed to combine the CmR backbone with Cas9. Three different cycles were run in an attempt to optimize the protocol; the following amounts were used in all three reactions:

_t Reactants Volume Q5 Master Mix 10.0 μL Cas9 DNA 5.0 μL CmR DNA 5.0 μL Nuclease Free H2O 0.00 μL Final Reaction Volume 20.0 μL

_t Thermocycler was varied for each of the three cPEC reactions as listed below:

_t cPEC1 cPEC2 cPEC3 Initial Denature 98°C/30 sec 98°C/30 sec 98°C/30 sec Denature 98°C/10 sec 98°C/20 sec 98°C/10 sec Anneal 56°C/30 sec 60°C/20 sec 66°C/30 sec Extend 72°C/90 sec 72°C/30 sec 72°C/90 sec

of cycles 10 15 10

Final Extension 72°C/10 min 72°C/10 min 72°C/10 min

_t

Nanodrop done on the three cPEC casC reactions with results listed below:

_t Sample ng/ μL 260/280 260/230 CasC1 489.3 1.80 0.72 CasC2 577.7 1.76 0.59 CasC3 542.5 1.80 0.74

_t

DNA cleanup done on these three casC cPEC reactions, followed by another nanodrop to examine purity with the results below.

_t Sample ng/ μL 260/280 260/230 CasC1 13.4 1.99 1.61 CasC2 11.1 2.02 1.15 CasC3* 9.9 1.84 1.21

_t

1% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 122V for ~50 min.

_t Well Loaded 1 2-Log DNA Ladder 2 cPEC1 3 cPEC2 4 cPEC3 5 GA Control 6 GA Positive Control 7 CasC 8 H2O Control

_t

Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.

_t Time 0 Time 1 Time 2 Time 3 Time 4 Time 5* Time 6 BWF+ 0.041 0.047 0.114 0.283 0.597 0.065 0.171 BWF+ w/cas9 0.035 0.046 0.110 0.262 0.530 0.059 0.154 _t

At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.

6/6/14

Plates with the Gibson Assembly products have shown very slow growth. Seven colonies are visible on the Gibson plate (shown with positive control plate below).

Above left: Gibson Assembly plate with 7 colonies. Above Right: Gibson Assembly positive control plate.

Week 5

6/9/14

Mini-preps were done following manufacturer's protocol except samples were centrifuged 4 minutes at the end (step 9). Ten samples were done that included 7 GA colonies, 1 GA control (labeled GA8), P50, and P51. All were subjected to a nanodrop and a restriction digestion with BamHI and EcoRV was performed on all the samples all except P51. The nanodrop results are shown below:

_t Sample ng/ μL 260/280 260/230 GA1 80.5 1.88 2.04 GA2 87.8 1.90 2.14 GA3 53.4 1.89 1.72 GA4 67.1 1.88 2.02 GA5 49.5 1.92 1.86 GA6 84.2 1.90 2.07 GA7 54.6 1.94 2.08 GA8 59.8 1.86 1.60 P50 91.3 1.88 2.12 P51 65.8 1.88 2.03 _t

Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.

_t Lane Sample 1 2-Log DNA Ladder 2 GA1 3 GA2 4 GA3 5 GA4 6 GA5 7 GA6 8 GA7 9 GA8 10 P50 Digest 11 SB1C3 12 Undigested GA1 13 Undigested GA2 14 Undigested GA3 15 Undigested P50 _t

Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ½ containing Kan and ½ containing Amp and Kan. The plates were made with increasing amounts of ATC.

gRNAs from cPEC were transformed and plated according to protocol with controls.

AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:

_t Reactants PCR AmilCP Original AmilCP Buffer 3 1.25 μL 1.25 μL BxsI 1.0 μL 2.5 μL DNA (125 ng) 4.0 μL 1.25 μL Nuclease Free H2O 6.25 μL 7.5 μL Final Reaction Volume 12.5 μL 12.5 μL _t 6/10/14

cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.

First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 μL of each to 40 μL nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 μL of each forward and reverse primer dimer that were added to 40 μL of N.F. H2O to produce a dilution (1:1:8). 5 μL of each primer mix was added to 25 μL of the Q5 master mix and 20 μL N.F. H2O to make a 50 μL total reaction for each sample.Products were checked by gel electrophoresis (shown below).

Next, a gel purification of a PCR P51 product was performed (to select for linear DNA). This product was digested with DpnI. A PCR cleanup was made of other P51 product that was not gel extracted. All of these steps were done following their recommended protocols.

Finally, cPEC reaction was done to combine the gel extracted P51 product with each of the gRNAs using the following amounts. The thermocycler set the same as on 6/3/14:

_t Reactants Volume Q5 Master Mix 25.0 μL Primer Mix 2.5 μL DNA (1:10 dilution) 1.0 μL Nuclease Free H2O 25.5 μL Final Reaction Volume 54.0 μL

_t

Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples: _t Sample ng/ μL 260/280 260/230 P51 416.9 1.82 0.67 AmilCP 430.5 1.82 0.67 158 472.2 1.80 0.73 756 479.6 1.78 0.73 666 475.8 1.80 0.73 729 485.4 1.79 0.75 SC1 464.6 1.80 0.73 SC2 475.1 1.80 0.74 _t

Titration Experiment: Done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. The plan is to make 50 mL of BWF+ w/cas9 and BWF+ w/o cas9 with a starting OD600 of 0.05. These will be placed in an incubator and allowed to grow until reaching 0.5. They will then be diluted again to 0.05 (in 50 mL). Serial dilutions will be made and plated on the prepared plates which will be incubated overnight.

Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.

_t Sample OD Amount added to 50 mL BWF+ 0.544 460 μL BWF+ w/cas9 0.556 450 μL _t

_t Time BWF+ BWF+ w/cas9 T0 0.059 0.058 T1 0.081 0.078 T2 0.180 0.168 T3 0.291 0.272 T4 0.398 0.366 T5 0.538 0.489 T6* 0.968 0.874 T7 0.165 0.153 T8 0.414 0.396 T9† 0.561 0.490 T10 0.057 0.056 _t

At T6, the cultures were again diluted down (5.17 mL BWF+, 5.72 mL BWF+ w/cas9 into 50 mL).

†After T9, 4.898 mL BWF+ and 4.278 mL BWF+ w/ cas9 were again diluted into 50 mL LB. A final OD was taken to ensure that the final OD was close to 0.05 before the cells were plated.

Cells were plated on the previously prepared plates containing increasing concentrations of ATC as previously described (6/9/14) and placed in 37°C incubator overnight.

6/11/14

Titration Experiment: Results

As seen from the images in the table below, the cas9 protein inhibits growth of the cells even prior to removal of repression by the TetR repressor, demonstrating some leaky expression. Increasing ATC concentrations results in increased cas9 expression in the cells as expected.

Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:

_t Sample ng/ μL 260/280 260/230 GE P51 13.7 1.94 1.18 GE AmilCP PCR 27.3 1.81 1.19 GE cas9 PCR 7.8 1.98 0.62 AmilCP DpnI digest 644.8 1.6 0.65 P50 PCR 493.9 1.8 0.73 GA1 MP 107.9 1.87 1.99 GA2 MP 144.9 1.88 2.11 GA3 MP 168.3 1.92 2.29 GA4 MP 131.1 1.91 1.93 GA5 MP 310.6 1.88 2.34 729-1 125.3 1.88 2.11 729-2 178.3 1.90 2.17 729-3 172.6 1.84 1.81 756-1 420.7 1.87 2.25 756-2 106.8 1.89 1.99

_t

6/12/14

Transformations done of all 6 gRNA cPEC reactions, P50 (+control), and P51 PCR (-control) Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).

_t Sample ng/ μL 260/280 260/230 Cas9 5.5 2.12 0.59

_t

P50 PCR amplification performed using the following amounts: - 25 μL Master Mix - 2.5 μL of forward primer (10 μM) - 2.5 μL of reverse primer (10 μM) - 1.0 μL DNA (1:50 dilution) - 19.0 μL NF H2O

Two samples prepared with different thermocycler settings as follows:

_t 1st Round 2nd Round Initial Denature 98°C/30 sec 98°C/2 min Denature 98°C/30 sec 98°C/30 sec Anneal 69°C/30 sec 68°C/30 sec Extend 72°C/3.5 min 72°C/5.5 min

of cycles 34 30

Final Extension 72°C/2 min 72°C/5 min _t Overnight cultures prepared on GA1-GA5, 729-1, 729-2, 729-3, and 759-1, 759-2 Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 μL Amp (50 ng/mL)

6/13/14

Mini-prep done on O/N cultures SC1-1 and SC1-2 followed by a nanodrop. Remainder of culture was spun down into a pellet and frozen.

_t Sample ng/ μL 260/280 260/230 SC1-1 30.7 2.02 2.06 SC1-2 32.5 1.94 1.48

_t

Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.

_t Sample Buffer 3 10X BSA Enzyme 1 Enzyme 2 Enzyme Mix* DNA (200 ng) MQ H2O Total Volume 756-1 2.0 μL 2.0 μL 0.5 μL EagI 0.5 μL XbaI -- 1.0 μL 14.0 μL 20.0 μL 756-2 2.0 μL 2.0 μL 0.5 μL EagI 0.5 μL XbaI -- 1.87 μL 13.13 μL 20.0 μL GA1 2.0 μL 2.0 μL -- -- 1.0 μL 1.85 μL 13.15 μL 20.0 μL GA2 2.0 μL 2.0 μL -- -- 1.0 μL 1.38 μL 13.62 μL 20.0 μL GA3 2.0 μL 2.0 μL -- -- 1.0 μL 1.19 μL 13.81 μL 20.0 μL GA4 2.0 μL 2.0 μL -- -- 1.0 μL 1.53 μL 13.47 μL 20.0 μL GA5 2.0 μL 2.0 μL -- -- 1.0 μL 0.64 μL 14.36 μL 20.0 μL P51 2.0 μL 2.0 μL -- -- 1.0 μL 4.0 μL 11.0 μL 20.0 μL Cas9 2.0 μL 2.0 μL -- -- 1.0 μL 2.0 μL 13.0 μL 20.0 μL _t

Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI

Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 756-1 3 756-2 4 P51 Digest 5 Sb1C3 6 GA1 7 GA2 8 GA3 9 GA4 10 GA5 11 Cas9 Digest _t

From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 μL sample plus 1 μL loading dye for each) as shown below: _t Lane Sample 1 2-Log DNA Ladder 2 GA4 3 P50 Digest 4 Sb1C3 5 CmR1 6 Cas9 PCR 7 Cas9 GE 8 GA4 MP _t

6/14/14

Gibson Assembly performed both with and without 2% DMSO. Each 20 μL reaction contained 10 μL Gibson Master Mix with the following as listed: Reaction #1 - 6.5 μL GE Cas9 - 3.5 μL GE AmilCP

Reaction #2 - 1 μL P50 PCR Product (1:10) - 5 μL GE AmilCP - 4 μL MQ H2O

Reaction #3 - 1 μL P50 PCR Product (1:10) - 5 μL GE AmilCP - 4 μL 1:10 dilution of 2% DMSO Bacterial Transformation: Done according to protocol - GA1-GA3 diluted 1:4 transformed with 2 μL (100 μL each) - BWF+ w/Cas9 from S.B. (25 μL full strength and 25 μL 1:10 dilution) - BWF+ (25 μL full strength and 25 μL 1:10 dilution) - NEB competent cells with P51 (50 μL) - Competent cells with P51 (50 μL) for comparison

Week 6

6/15/14

GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ μL Amp.

6/16/14

Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:

_t Sample ng/ μL 260/280 260/230 1-1 17.5 2.08 1.94 1-2 54.1 1.93 1.96 1-3 30.4 2.00 1.78 2-1 45.8 1.93 1.90 2-2 45.4 1.92 1.89 2-3 46.9 1.93 1.97 2-4 53.5 1.96 2.01 2-5 53.5 1.94 2.04 2-6 41.9 1.95 1.84 2-7 59.3 1.94 1.99 2-8 46.5 1.94 1.98

_t

Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 μL and MQ H2O was not added). - 1.0 μL Buffer D - 3.0 μL Enzyme - 3.0 μL sample DNA - 3.0 μL MQ H2O - Total Reaction Volume = 10 μL

Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 1-1 3 1-2 4 1-3 5 2-1 6 2-2 7 2-3 8 2-4 9 2-5 10 2-6 11 2-7 12 2-8 _t

Repeated digestion (protocol above) on select samples to run again for more definition on gel. Samples are run on a 1% agarose gel. The following lists the sample loaded into each lane.

_t Lane Sample 1 2-Log DNA Ladder 2 1-1 3 1-2 4 2-1 5 sb1c3 6 Digested sb1c3 7 Digested p50 8 2-Log DNA Ladder _t

Bacterial Transformation: Done according to protocol

- 7 CPEC products including p51 as the negative control (10 μL 1:4 dilution) with 40 μL DH5α - BWF+ w (40μL)/ S.B. cas9 (1 μL) - BWF+ (1 μL)

Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen

6/17/14

_t Sample Buffer Enzyme 1 EnzymeMix* DNA (200 ng) MQ H2O Total Volume GA x 1-1 1.0 μL cutsmart 3.0 μL XbaI (1:50) -- 3.0 μL 3.0 μL 10.0 μL GA x 1-2 1.0 μL cutsmart 3.0 μL XbaI (1:50) -- 3.0 μL 3.0 μL 10.0 μL GA x 2-1 1.0 μL cutsmart 3.0 μL XbaI (1:50) -- 3.0 μL 3.0 μL 10.0 μL p50 linear 1.0 μL cutsmart 3.0 μL SepI (1:50) -- 3.0 μL 3.0 μL 10.0 μL GA H 1-1 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL GA H 1-2 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL GA H 2-1 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL p50 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL _t

Enzyme mix: 1:1:23 dilution of xhoI and HindIII

Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 GA x 1-1 3 GA x 1-2 4 GA x 2-1 5 p50 6 sb1c3 7 GA H 1-1 8 GA H 1-2 9 GA H 2-1 10 p50 11 2-Log DNA Ladder

_t

6/18/14

Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips were then ejected into 3ml LB+AMP for O/N culture.The following was added to each tube to make a 25 μL reaction. - 12.5 μL 2X Master Mix - 1.25 μL of forward primer (10 μM) - 1.25 μL of reverse primer (10 μM) - 10.0 μL MQ H2O

For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).

_t Initial Denature 98°C/10 min Denature 98°C/30 sec Anneal 65°C/30 sec Extend 72°C/30 sec

of cycles 35

Final Extension 72°C/5 min _t 6/19/14 Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.

Week 7

6/23/14 Chemically competent cells of SBcas9 • BWF+. Done according to protocol.

6/25/14 Mini prep done to protocol on CPEC products.

Nanodrops were taken to assess the concentration and purity of the samples: _t Sample ng/ μL 260/280 260/230 2H 65.2 1.88 2.21

    3E	56.3	1.91	2.01

4E 38.9 1.93 2.22 5B 54.1 1.86 1.54 6B 69.4 1.88 2.12 7B 66.6 1.82 1.56 8B 69.7 1.83 1.75 9B 61.1 1.89 1.81 10C 52.2 1.86 1.65 11B 43.9 1.86 1.33 12B 57.5 1.83 1.53 _t

Sequencing 12 CPEC products: 12.5 μL DNA + 2.5 μL 10μm primer (CRE22)

_t CPEC product 1 1A 2 1C 3 2B 4 2C 5 3B 6 3H 7 4C 8 4D 9 5E 10 5F 11 6C 12 6F _t

6/26/14 Sequencing: Total volume 0f 15 μL _t CPEC Product DNA μL Primer CRE22 MQ H2O 1 2B 12.5μL 2.5 μL -- 2 2C 12.0μL 2.5μL 0.5μL 3 3B 10.3μL 2.5μL 2.2μL 4 3H 11μL 2.5μL 1.5μL 5 4E 12.5μL 2.5μL -- 6 5E 11.3μL 2.5μL 1.2μL 7 11B 12.5μL 2.5μL -- 8 2H 8μL 2.5μL 4.5μL 9 6B 8μL 2.5μL 4.5μL 10 7B 8μL 2.5μL 4.5μL 11 8B 8μL 2.5μL 4.5μL 12 9B 8μL 2.5μL 4.5μL 13 4C 8μL 2.5μL 4.5μL 14 3E 10μL 2.5μL 2.5μL 15 5B 10μL 2.5μL 2.5μL 16 10C 10μL 2.5μL 2.5μL 17 12B 10μL 2.5μL 2.5μL

_t

Bacterial Transformation of SBcas9• ER: Done according to protocol.

6/27/14 Mini prep done to protocol on CPEC products. Nanodrops were taken to assess the concentration and purity of the samples: _t Sample ng/ μL 260/280 260/230 1A 66.9 1.90 2.26

    1C	61.3	1.92	2.20

2B 57.8 1.91 2.27 3B 69.4 1.88 1.14 3H 51.7 1.89 2.24 9B 33.6 1.99 1.62 10C 49.8 1.93 2.09 11B 40.5 1.94 2.01 12B 51.9 1.89 1.17 _t

Sequenced all of the above products (total volume of 15 μL: 10 μL DNA+ 2.5 μLprimer (CRE22)+2 .5 μL MQ H2O Exceptions: -9B received 12.5 μL DNA + 2.5 μL primer (CRE22) -12B received p51 seq1 as the primer

Glycerol stock of all the above samples to protocol: 1:1 w/ 40% glycerol for a total volume of 500 μL each.

Positive sequencing results: 1A, 1C, 2B, 3B, 3H, 9B, 10C, 11B, 12B

Bacterial Transformation of SBcas9• ER and PUC19 as the positive control: Done according to protocol.

Prepared 5mL O/N cultures in LB+AMP of colonies selected from glycerol stocks

Mini preps done to protocol on the below samples Nanodrops were taken to assess the concentration and purity of the samples: _t Sample ng/ μL 260/280 260/230 1A 78.7 1.90 2.08

    1C	54.3	1.88	1.76

2B 335.4 1.48 0.91 3B 52.1 1.91 1.96 3H 59.7 1.89 2.03 _t

Week 8

6/30/14 Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51: Done according to protocol.

7/1/14

Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction. _t Sample ng/ μL p50 Amplicon 27.3

    GE AmilCP	493.9

_t

Gel extracted

cPEC performed to combine the p50 amplicon with AmilCP following the cPEC protocol and using the following amounts:

_t Reactants Volume Q5 Master Mix 12.5 μL Insert (AmilCP) 5 μL Template DNA p50 2.0 μL (1:20 dilution) 25% DMSO 2.0 μL Nuclease Free H2O 3.5 μL Final Reaction Volume 25.0 μL _t

Thermocycler conditions:

_t cPEC Initial Denature 98°C/30 sec Denature 98°C/10 sec Anneal 60°C/30 sec Extend 72°C/105 sec

of cycles 10

Final Extension 72°C/10 min _t

Successful Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51 (from 6/30) . O/N cultures of each were then made.

7/4/14 Electroporation of p50 amplicon • AmilCP with Marcello 7/7/14 Electroporation was unsuccessful. Redone using Bacterial Transformation done to protocol. Primer design for qPCR Streaked p50 on plate Week 9 7/8/14 mini prepped p50 done to protocol Colony PCR done to protocol on the p50+AmilCP transformation product . Selected 12 colonies. *PCR tube lids opened in thermocycler for samples: 8, 12, and positive control Colony PCR products are run on a 1% agarose gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 1 3 2 4 3 5 4 6 5 7 6 8 7 9 8 10 9 11 10 12 11 13 12 14 (+) control 15 (-) control _t

7/9/14 Mini prepped colony PCR samples: 1,7,8,10,11, and p50 Followed by nanodrop with results listed below:

_t Sample ng/ μL 260/280 260/230 1 89 1.90 2.22 7 110.9 1.88 2.22 8 134.4 1.85 1.77 10 140.9 1.87 2.10 11 93.6 1.89 2.21 p50 191.7 1.87 2.35 _t

The above samples (except 8) were sent for sequencing. Total volume 0f 15 μL _t Product DNA μL Primer CRE22 MQ H2O 1 1 6.0μL 5.0 μL 4.0μL 2 7 5.0μL 5.0μL 5.0μL 3 10 4.0μL 5.0μL 6.0μL 4 11 6.0μL 5.0μL 4.0μL _t

Transformation done to protocol on all gRNAs into BWF+

7/10/14 Site directed Mutagenesis (SDM) performed on original p50, 1, 7, 10, 11 to remove Xba1 from p50 Primer names: p50TetRXba1F and p50TetRXba1R Primer Mix: 1 μL forward + 1 μL reverse + 18 μL MQ H2O

PCR run on the following samples of p50: p50 DNA dilution for original, 1,7,10, and 11 p50 DNA sample original- 1:38 dilution p50 DNA sample 1- 1:18 dilution p50 DNA sample 7- 1:22 dilution p50 DNA sample 10- 1:28 dilution p50 DNA sample 11- 1:19 dilution

_t Reactants Volume Q5 Master Mix 12.5 μL 10 μM primer mix 2.5 μL DNA p50 2.0 μL Nuclease Free H2O 8.0 μL Final Reaction Volume 25.0 μL _t

Thermocycler conditions:

_t Initial Denature 98°C/30 sec Denature 98°C/30 sec Anneal 55°C/30 sec Extend 72°C/3.5 minutes

of cycles 15 cycles

Final Extension 72°C/5 minutes _t SDM performed on 6 gRNAs and the original p51 to remove spe1 site

PCR preformed: _t Reactants Volume Q5 Master Mix 12.5 μL 10 μM primer mix 2.5 μL DNA p51 2.0 μL Nuclease Free H2O 8.0 μL Final Reaction Volume 25.0 μL _t

primer mix: 1 μL forward primer+ 1 μL reverse primer +18 μL MQH2O. Primers varied for each gRNA
control consisted of DNA only, no primers

Thermocycler conditions:

_t Initial Denature 98 95°C/30 sec Denature 98 95°C/30 sec Anneal 55 55°C/1min Extend 72 68°C/3min

of cycles 15 cycles

Final Extension 72 3 min _t

7/11/2014

All PCR gRNAs DPN1 digested: 1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction below the mineral oil overlay. 2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA.

Transformation done to protocol for all gRNAs into DH5α. Full volume (250μl) plated onto Amp plates and incubated o/n. Week 10 7/16 O/N cultures of SDM gRNA p51 (4 colonies) and SC1 (2 colonies) 7/17 Mini prepped all p51 and SC1 cultures to protocol and then nano dropped:

_t Sample ng/ μL 260/280 260/230 p51 (1) 62.5 1.90 1.65

   p51(2)	87.2	1.95	2.20

p51(3) 112.7 1.81 1.34 p51(4) 67.1 1.92 2.24 sc1 (1) 126.5 1.83 1.48 sc1(2) 101.9 1.88 1.84 _t

Restriction digest done to protocol:

_t Sample Buffer Green Fast Digest Enzyme Mix* DNA (200 ng) MQ H2O Total Volume p51 (1) 1.0 μL 1.0 μL 3.0 μL 5.0 μL 10.0 μL p51 (2) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL p51 (3) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL p51 (4) 1.0 μL 1.0 μL 3.0 μL 5.0 μL 10.0 μL SC1 (1) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL SC1 (2) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL _t

Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL BgI1 + 23.0 μL mqH2O

Samples evaporated after digesting to a total volume of around 4 μL

Run on a 1% Gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 SDM p51 (1) 3 SDM p51 (3) 4 SDM SC1 (1) 5 SDM SC1 (2) 6 Blank 7 SDM P51 (4) _t

7/19/2014 PCR amplify p50

_t Reactants Volume Q5 Master Mix 12.5 μL 10 μM forward primer 1.5 μL 10 μM reverse primer 1.5 μL DNA p50 (1:50) 1.0 μL Nuclease Free H2O 9.0 μL Final Reaction Volume 25.0 μL _t 7/21/2014

Restriction digest same gRNAs again

_t Sample Buffer cutsmart Enzyme Xbal Enzyme Spe1 DNA (200 ng) MQ H2O Total Volume p51 (1) 2.0 μL 1.0 μL 1.0 μL 3.2 μL 12.8 μL 20.0 μL p51 (2) 2.0 μL 1.0 μL 1.0 μL 2.3 μL 13.7 μL 20.0 μL p51 (3) 2.0 μL 1.0 μL 1.0 μL 1.8 μL 14.2 μL 20.0 μL p51 (4) 2.0 μL 1.0 μL 1.0 μL 3.0 μL 13.0μL 20.0 μL SC1 (1) 2.0 μL 1.0 μL 1.0 μL 1.6 μL 14.4 μL 20.0 μL SC1 (2) 2.0 μL 1.0 μL 1.0 μL 2.0 μL 14.0μL 20.0 μL P51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL P51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL p51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL _t

Enzymes are used as a 1:25 dilution

Run on a 1% Gel. The results were still unclear, the following day samples were sent for sequencing.

Week 11

7/23/2014 Sent p51 SDM 1, 3, 4 and p51 original as a control for sequencing. Confirmation that SDM was successful and p51 no longer has the restriction site spe1 in the gRNA location.

Sequencing: Total volume 0f 15 μL _t SDM Product DNA μL Primer p51seq5 MQ H2O 1 SDM1p51 8.0μL 2.5 μL 4.5μL 2 SDM3p51 4.4μL 2.5μL 8.1μL 3 SDM4p51 7.5μL 2.5μL 5.0μL 4 p51 control 4.2μL 2.5μL 8.3μL _t

Mini prepped 10 o/n cultures of SDM of p50. Done to protocol. Glycerol stocks make of all 10 cultures. Mini prepped previous gibson colonies 1 and 2. SDM of p51 for all samples were confirmed

Nanodrops were taken to assess the concentration and purity of the samples:

_t Sample ng/ μL 260/280 260/230 p50 123.7 1.84 2.15

    SDM1	179.6	1.88	2.19

SDM2 117.4 1.90 2.21 SDM3 140.4 1.84 1.81

   SDM4	162.6	1.89	2.22

SDM5 117.3 1.89 2.00 SDM6 176.4 1.88 2.01 SDM7 177.0 1.87 2.30 SDM8 250.1 1.89 2.25 SDM9 298.3 1.90 2.31 SDM10 136.0 1.91 2.17 Gibson1 110.8 1.89 2.14 Gibson2 160.3 1.87 2.15

_t

Restriction digest done to protocol:

_t Sample Buffer Cutsmart Enzyme Mix* DNA (200ng) MQ H2O Total Volume SDM p50 (1) 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL SDM p50 (2) 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL SDM p50 (3) 2.0 μL 1.0 μL 1.4 μL 15.6 μL 20.0 μL SDM p50 (4) 2.0 μL 1.0 μL 1.2 μL 15.8μL 20.0 μL SDM p50 (5) 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL SDM p50 (6) 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL SDM p50 (7) 2.0 μL 1.0 μL 1.2 μL 15.8μL 20.0 μL SDM p50 (8) 2.0 μL 1.0 μL 1.0 μL 16.0μL 20.0 μL SDM p50 (9) 2.0 μL 1.0 μL 1.0 μL 16.0μL 20.0 μL SDM p50 (10) 2.0 μL 1.0 μL 1.5 μL 15.5μL 20.0 μL p50control 2.0 μL 1.0 μL 2.0 μL 15.0μL 20.0 μL _t

Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL Xba1 + 48.0 μL mqH2O

Restriction Digest for gibson done to protocol: _t Sample Buffer D Enzyme EcoRV DNA (200 ng) MQ H2O Total Volume Gibson 1 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL Gibson 2 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL _t

EcoRv dilution (1:25)

Run on a 1% Gel. The following lists the sample loaded into each lane. _t Lane Sample 1 2-Log DNA Ladder 2 SDM p50 (1) 3 SDM p50 (2) 4 SDM p50 (3) 5 SDM p50 (4) 6 SDM p50 (5) 7 SDM p50 (6) 8 SDM p50 (7) 9 SDM p50 (8) 10 SDM p50 (9) 11 SDM p50 (10) 12 p50 control 13 p50 undigested 14 Gibson 1 15 Gibson 2 16 sb1c3 17 blank

_t

7/25/2014

Restriction digest done to protocol:

_t Sample Buffer Cutsmart Enzyme Mix* DNA (250 ng) MQ H2O Total Volume SDM p50 (1) 2.0 μL 1.0 μL 1.5 μL 20.0 μL 25.0 μL SDM p50 (2) 2.0 μL 1.0 μL 2.0 μL 19.5 μL 25.0 μL SDM p50 (3) 2.0 μL 1.0 μL 1.8 μL 19.7 μL 25.0 μL SDM p50 (4) 2.0 μL 1.0 μL 1.5 μL 20.0μL 25.0 μL SDM p50 (5) 2.0 μL 1.0 μL 2.0 μL 19.5 μL 25.0 μL SDM p50 (6) 2.0 μL 1.0 μL 1.5 μL 20.0 μL 25.0 μL SDM p50 (10) 2.0 μL 1.0 μL 2.0 μL 19.5μL 25.0 μL p50control 2.0 μL 1.0 μL 2.0 μL 19.5μL 25.0 μL _t

Enzyme mix: 5.5μL Xba1 + 5.5μL Spe1 for a total volume of 11μL

Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.

Restriction Ligation

_t Sample Buffer Cutsmart EcoR1 Xba1 Spe1 Pst1 DNA (200 ng) MQ H2O Total Volume SDM p50 (1) 5.0 μL -- 1.0 μL -- 1.0 μL 1.0 μL 42.0 μL 50.0 μL SDM p50 (4) 5.0 μL -- 1.0 μL -- 1.0 μL 1.0 μL 42.0 μL 50.0 μL SDM p50 (6) 5.0 μL -- 1.0 μL -- 1.0 μL 1.0 μL 42.0 μL 50.0 μL Litimus28i 5.0 μL -- -- 1.0 μL 1.0 μL 1.5 μL 41.5μL 50.0 μL Litimus+Ic3 5.0 μL 1.0 μL -- -- 1.0 μL 0.8 μL 42.2 μL 50.0 μL PsBGA1 (RFP) 5.0 μL 1.0 μL -- 1.0 μL -- 3.8 μL 39.2 μL 50.0 μL _t

Sequenced SDMp50 was not the correct product, the above project did not continue.

8/18/2014 O/N cultures of MG, Bw+Kan, Bw-kan

8/19/2014 O/N cultures were all made competent according to protocol O/D recordings:

_t Time MG Bw+kan Bw(-)kan 11:00 .042 .026 .071 11:34 .086 .035 .088 12:10 .239 .078 .223 12:20 .328 .098 .296 12:28 -- -- .325 12:47 -- .196 -- 1:10 -- .308 -- 1:15 -- .320 -- _t

The cells were all pulled at the time they were at an O/D nearest .325. All O/D’s were diluted to .30 Once the cells were made competent they were transformed using a gRNA target, gRNA scramble, and a sb1c3 strain.

@@ Line 1: / Line 1: @@
-{{Template:UCB-Main}}
-{{UCB-NavBar}}
-__FORCETOC__
 Week 1
 /12/14
@@ Line 13: / Line 10: @@
 Glycerol stocks
 -	Stocks made from overnight cultures (3 from each)
--	500 ?L bacteria + 500 ?L 40% glycerol stock
+-	500 μL bacteria + 500 μL 40% glycerol stock
 *Glass tube containing #P44250 broke and was discarded – overnight repeated
@@ Line 23: / Line 20: @@
 /14/14
-{| class = "wikitable"
+_t Nano Drop of Mini Prep
-|-
+Sample	260/280 Ratio	260/230 Ratio	DNA Concentration
-!  Nano Drop of Mini Prep
+P44250	1.94	2.17	49.4 ng/μL
-!
+P44251	1.94	2.47	52.7 ng/μL
-!
+_t
-!
-|-
-| Sample
-| 260/280 Ratio
-| 260/230 Ratio
-| DNA Concentration
-|-
-| P44250
-| 1.94
-| 2.17
-| 49.4 ng/?L
-|-
-| P44251
-| 1.94
-| 2.47
-| 52.7 ng/?L
-|-
-|}
 *Samples were blanked with EB Buffer
@@ Line 59: / Line 34: @@
-{| class = "wikitable"
+_t	P44250	P44251
-|-
+Cutsmart Buffer	5.0 μL	5.0 μL
-!
+XbaI	1.0 μL	1.0 μL
-! P44250
+XhoI	1.0 μL	--
-! P44251
+SpeI	1.0 μL	--
+EcoRI-HF	--	1.0 μL
-|-
+DNA (200 ng)	4.0 μL	4.0 μL
-| Cutsmart Buffer
+MQ H2O	38.0 μL	39.0 μL
-| 5.0 ?L
+Total Volume	50.0 μL	50.0 μL
-| 5.0 ?L
+_t
-|-
-| XbaI
-| 1.0 ?L
-| 1.0 ?L
-|-
-| XhoI
-| 1.0 ?L
-| --
-|-
-| SpeI
-| 1.0 ?L
-| --
-|-
-| EcoRI-HF
-| --
-| 1.0 ?L
-|-
-| DNA (200 ng)
-| 4.0 ?L
-| 4.0 ?L
-|-
-| MQ H2O
-| 38.0 ?L
-| 39.0 ?L
-|-
-| Total Volume
-| 50.0 ?L
-| 50.0 ?L
-|-
-|}
 Agarose Gel Electrophoresis
 -1% agarose gel prepared
--2 ?L loading dye added to 18 ?L of each sample for loading into each lane
+-2 μL loading dye added to 18 μL of each sample for loading into each lane
 -1Kb+ DNA Ladder used
 -Overnight staining in EtBr done to better visualize faint bands
@@ Line 126: / Line 63: @@
 Plate Streaking
 -Used to check the TetR gene in the P44250 plasmid
--2 mL LB containing 50 mg/ ?L Tet added to plate and allowed to soak in
+-2 mL LB containing 50 mg/ μL Tet added to plate and allowed to soak in
 -Plates left at room temperature overnight
@@ Line 158: / Line 95: @@
 /19/14
 Transformation:
-?L P44250 (cas9) plasmid added to K12 E. coli strain
+μL P44250 (cas9) plasmid added to K12 E. coli strain
 Heat shock done according to protocol (45 sec @ 42°C)
 Cultures incubated at 37°C for 1.5 hours
 Plates with K12 and the plasmid were plated by streaking as follows:
--	Amp, Amp (? plate with 1:10 dilution, ? plate 1:1), Amp + Kan, Amp + Kan (? plate with 1:10 dilution, ? plate 1:1)
+-	Amp, Amp (½ plate with 1:10 dilution, ½ plate 1:1), Amp + Kan, Amp + Kan (½ plate with 1:10 dilution, ½ plate 1:1)
 /20/14
 Plates from 5/19/14 (shown below).
@@ Line 173: / Line 110: @@
 /22/14
 Transformation: (Chemically competent BWF+ cells with P44250 plasmid)
-?L P44250 (cas9) plasmid added to conjugated BWF+ strain
+μL P44250 (cas9) plasmid added to conjugated BWF+ strain
 Transformation protocol followed with following exceptions:
--200 ?L SOC added to each sample
+-200 μL SOC added to each sample
 -Incubated for 2 hours
--1:10 dilution made (10 ?L into 90 ?L SOC)
+-1:10 dilution made (10 μL into 90 μL SOC)
 -Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution
@@ Line 183: / Line 120: @@
 /23/14
 Arrival of primers previously ordered
-	All were reconstituted in MQ H2O to 100 ?M concentration
+	All were reconstituted in MQ H2O to 100 μM concentration
 Results of plates from 5/22/14:
@@ Line 194: / Line 131: @@
-Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 ?L liquid culture overnight with the following antibiotics:
+Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 μL liquid culture overnight with the following antibiotics:
 -	AMP
 -	Kan
@@ Line 207: / Line 144: @@
 PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid
-	*DNA samples diluted so each contains 0.5 ng/?L
+	*DNA samples diluted so each contains 0.5 ng/μL
-{| class = "wikitable"
+_t	Cas9	CMR
-|-
+Q5 Master Mix volume	25 μL	25 μL
-!
+dcas9 Primer volume	2.5 μL	--
-! Cas9
+dCMR Primer volume	--	2.5 μL
-! CMR
+DNA added	2.5 μL	2.5 μL
+Q5 Polymerase volume	0.5 μL	0.5 μL
-|-
+Nuclease Free H2O	20 μL	20 μL
-| Q5 Master Mix volume
+Final Reaction Volume	50.0 μL	50.0 μL
-| 25 ?L
+_t
-| 25 ?L
-|-
-| dcas9 Primer volume
-| 2.5 ?L
-| --
-|-
-| dCMR Primer volume
-| --
-| 2.5 ?L
-|-
-| DNA added
-| 2.5 ?L
-| 2.5 ?L
-|-
-| Q5 Polymerase volume
-| 0.5 ?L
-| 0.5 ?L
-|-
-| Nuclease Free H2O
-| 20 ?L
-| 20 ?L
-|-
-| Final Reaction Volume
-| 50.0 ?L
-| 50.0 ?L
-|-
-|}
 PCR Settings: 30 cycles as follows
 -	98 °C Denature 30 sec
@@ Line 260: / Line 163: @@
 /25/14
-Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 ?L EtBr.
+Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 μL EtBr.
-	Lane 1: 5 ?L 2-Log DNA Ladder
+	Lane 1: 5 μL 2-Log DNA Ladder
 	Lane 2: CMR sample* (~2 Kb size)
 	Lane 3: Cas9 sample* (~5 Kb size)
-*Each contained 8 ?L reaction sample + 2 ?L loading dye
+*Each contained 8 μL reaction sample + 2 μL loading dye
 Gel Results:
@@ Line 278: / Line 181: @@
 /27/14
 Repeat of PCR using same protocol as listed on 5/24/14 except for different times on the thermocycler as follows:
-{| class = "wikitable"
+_t	Cas9	CMR
-|-
+Initial Denature	98°C/2 min	98°C/2 min
-!
+Denature	98°C/30 sec	98°C/30 sec
-! Cas9
+Anneal	69°C/30 sec	72°C/30 sec
-! CMR
+Extend	69°C/ 2.5 min	72°C/2.5 min
+Final Extension	72°C/6 min	72°C/5 min
-|-
+_t
-| Initial Denature
-| 98°C/2 min
-| 98°C/2 min
-|-
-| Denature
-| 98°C/30 sec
-| 98°C/30 sec
-|-
-| Anneal
-| 69°C/30 sec
-| 72°C/30 sec
-|-
-| Extend
-| 69°C/ 2.5 min
-| 72°C/2.5 min
-|-
-| Final Extension
-| 72°C/6 min
-| 72°C/5 min
-|-
-|}
 * Thermocycler containing CMR stopped after 1 cycle and held at 98°C for ~3hours. Machine was reset and run. This sample may no longer be viable.
@@ Line 317: / Line 194: @@
 Dry lab – primer designs
 Repeat of PCR for the CMR reaction, same as described on 5/24/14 and shown below.
-{| class = "wikitable"
+_t Reactants	CMR
-|-
+Q5 Master Mix 	25 μL
-!  Reactants
+dCMR Primer 	2.5 μL
-! CMR
+Q5 Polymerase 	0.5 μL
+CMR DNA	2.0 μL
-|-
+Nuclease Free H2O	20 μL
-| Q5 Master Mix
+Final Reaction Volume	50.0 μL
-| 25 ?L
+_t
-|-
-| dCMR Primer
-| 2.5 ?L
-|-
-| Q5 Polymerase
-| 0.5 ?L
-|-
-| CMR DNA
-| 2.0 ?L
-|-
-| Nuclease Free H2O
-| 20 ?L
-|-
-| Final Reaction Volume
-| 50.0 ?L
-|-
-|}
 /29/14
 Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.
-{| class = "wikitable"
+_t Lane	Sample	Amount	Loading dye amount
-|-
+	2-Log DNA Ladder	0.5  μL	--
-!  Lane
+	Cas9	5.0  μL	1.0  μL
-! Sample
+	CMR 1 (from 5/24)	5.0  μL	1.0  μL
-! Amount
+	CMR 2 (from 5/27)	5.0  μL	1.0  μL
-! Loading dye amount
+	CMR 3 (from 5/28)	5.0  μL	1.0  μL
+_t
-|-
-| 1
-| 2-Log DNA Ladder
-| 0.5  ?L
-| --
-|-
-| 2
-| Cas9
-| 5.0  ?L
-| 1.0  ?L
-|-
-| 3
-| CMR 1 (from 5/24)
-| 5.0  ?L
-| 1.0  ?L
-|-
-| 4
-| CMR 2 (from 5/27)
-| 5.0  ?L
-| 1.0  ?L
-|-
-| 5
-| CMR 3 (from 5/28)
-| 5.0  ?L
-| 1.0  ?L
-|-
-|}
 Results from Gel (shown below):
 	Lane 2: Cas9 band visible of appropriate size (approximately 5,000 bp)
@@ Line 400: / Line 222: @@
 Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following  the protocol. Following amounts:
--	Cas9: 110 ?L buffer with 0.1103 g sample
+-	Cas9: 110 μL buffer with 0.1103 g sample
--	CMR1: 53 ?L buffer with 0.0533 g sample
+-	CMR1: 53 μL buffer with 0.0533 g sample
--	CMR2: 90 ?L buffer with 0.0904 g sample
+-	CMR2: 90 μL buffer with 0.0904 g sample
 Nanodrops performed to determine DNA concentration and purity with the following results:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230	**ng/ μL	**260/280	**260/230
-|-
+Gel-Cas9	7.3	2.20	0.57	5.2	2.06	0.52
-!  Sample
+Cas9	483.2	1.78	0.72	36.3	1.85	1.72
-! ng/ ?L
+Gel-CMR1	9.6	2.02	0.63	6.5	2.35	0.90
-! 260/280
+Gel-CMR2	8.3	2.06	0.83	5.0	2.21	0.69
-! 260/230
+CMR2	618.8	1.66	0.61	44.5	1.89	1.41
-! **ng/ ?L
+CMR 3	535.8	1.73	0.61	17.8	1.89	1.31
-! **260/280
+CMR1	516.6	1.69	0.61	68.5	1.84	1.74
-! **260/230
+_t
-|-
-| Gel-Cas9
-| 7.3
-| 2.20
-| 0.57
-| 5.2
-| 2.06
-| 0.52
-|-
-| Cas9
-| 483.2
-| 1.78
-| 0.72
-| 36.3
-| 1.85
-| 1.72
-|-
-| Gel-CMR1
-| 9.6
-| 2.02
-| 0.63
-| 6.5
-| 2.35
-| 0.90
-|-
-| Gel-CMR2
-| 8.3
-| 2.06
-| 0.83
-| 5.0
-| 2.21
-| 0.69
-|-
-| CMR2
-| 618.8
-| 1.66
-| 0.61
-| 44.5
-| 1.89
-| 1.41
-|-
-| CMR 3
-| 535.8
-| 1.73
-| 0.61
-| 17.8
-| 1.89
-| 1.31
-|-
-| CMR1
-| 516.6
-| 1.69
-| 0.61
-| 68.5
-| 1.84
-| 1.74
-|-
-|}
 *Trial 2 completed on 5/30/14 shown in these columns
 Values for the ratios should be between 2.0 and 2.2 for a pure sample. The values for Trial #1 are much too low. In order to increase sample purity, a DNA cleanup is performed following the protocol given in the E.Z.N.A. gel extraction kit in amounts shown below.
--	25 ?L samples
+-	25 μL samples
--	25 ?L binding buffer
+-	25 μL binding buffer
--	Washed 2x with 700 ?L SPW
+-	Washed 2x with 700 μL SPW
--	30 ?L elution buffer added, allowed to rest 4 minutes
+-	30 μL elution buffer added, allowed to rest 4 minutes
 -	Centrifuged on max for 2 min
@@ Line 496: / Line 252: @@
 Restriction Digestion performed on Cas9 and CMR1 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
-?L sample
+μL sample
-.0	?L DPN1 (100 fold dilution)
+.0	μL DPN1 (100 fold dilution)
-.0 ?L 10X CutSmart Buffer
+.0 μL 10X CutSmart Buffer
 Gibson Assembly performed to join pSB1C3 backbone with cas9 PCR product. Protocol for Gibson was followed for the reaction.
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Gibson Master Mix 	10 μL
-!  Reactants
+Cas9	1.6 μL (50 ng)
-! Volume
+Cmr	2.4 μL (143 ng)
+Nuclease Free H2O	6 μL
-|-
+Final Reaction Volume	20.0 μL
-| Gibson Master Mix
+_t
-| 10 ?L
-|-
-| Cas9
-| 1.6 ?L (50 ng)
-|-
-| Cmr
-| 2.4 ?L (143 ng)
-|-
-| Nuclease Free H2O
-| 6 ?L
-|-
-| Final Reaction Volume
-| 20.0 ?L
-|-
-|}
 Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.
@@ Line 535: / Line 271: @@
 /2/14
-Grow Gibson colonies in 6 mL LB media + 30 ?L chloroamp
+Grow Gibson colonies in 6 mL LB media + 30 μL chloroamp
 Transform Gibson colonies following protocol and with amounts as shown:
--	BWF+ w/cas9:	15 ?L into 135 ?L SOC
+-	BWF+ w/cas9:	15 μL into 135 μL SOC
--	BW w/cas9:	15 ?L into 135 ?L SOC
+-	BW w/cas9:	15 μL into 135 μL SOC
--	BWF+ (no cas9):  30 ?L into 270 ?L SOC
+-	BWF+ (no cas9):  30 μL into 270 μL SOC
--	BW (no cas9): 30 ?L into 270 ?L SOC
+-	BW (no cas9): 30 μL into 270 μL SOC
-?L of the above four reactions plated as follows and incubated:
+μL of the above four reactions plated as follows and incubated:
 -	BWF+ w/cas9:	Amp + Kan
 -	BW w/cas9:	Amp + Kan
@@ Line 559: / Line 295: @@
 NanoPrep on GAcasC
-{| class = "wikitable"
+_t ng/ μL	260/280	260/230
-|-
+.0	1.88	2.24
-!  ng/ ?L
+_t
-! 260/280
-! 260/230
-|-
-| 201.0
-| 1.88
-| 2.24
-|-
-|}
 Digest performed according to protocol on GAcasC and P50 plasmid using EcoRV and BamHI as follows:
-{| class = "wikitable"
+_t Reactants	GAcasC	P50
-|-
+Buffer 3	2.5 μL	2.5 μL
-!  Reactants
+EcoRV	0.5 μL	0.5 μL
-! GAcasC
+BamHI	0.5 μL	0.5 μL
-! P50
+BSA (1:4 dilution) 	1.0 μL	1.0 μL
+DNA (250 ng)	1.25 μL	5.0 μL
-|-
+MQ H2O	19.25 μL	15.5 μL
-| Buffer 3
+Total Volume	25.0 μL	25.0 μL
-| 2.5 ?L
+_t
-| 2.5 ?L
-|-
-| EcoRV
-| 0.5 ?L
-| 0.5 ?L
-|-
-| BamHI
-| 0.5 ?L
-| 0.5 ?L
-|-
-| BSA (1:4 dilution)
-| 1.0 ?L
-| 1.0 ?L
-|-
-| DNA (250 ng)
-| 1.25 ?L
-| 5.0 ?L
-|-
-| MQ H2O
-| 19.25 ?L
-| 15.5 ?L
-|-
-| Total Volume
-| 25.0 ?L
-| 25.0 ?L
-|-
-|}
-% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 120V for ~1 hour.
-{| class = "wikitable"
-|-
-!  Well
-! Loaded
-|-
-| 1
-| DNA Ladder
-|-
-| 2
-| GAcasC digest
-|-
-| 3
-| CmR1 Dpn1 product
-|-
-| 4
-| Cas9 Dpn1 product
-|-
+% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 120V for ~1 hour.
-| 5
-| P50 digestion product
-|-
+_t Well	Loaded
-|}
+	DNA Ladder
+	GAcasC digest
+	CmR1 Dpn1 product
+	Cas9 Dpn1 product
+	P50 digestion product
+_t
 Gel Results:
 	Lane 2 should have multiple products which are not present in the gel. The other lanes are as expected.
@@ Line 655: / Line 327: @@
 PCR using Q5 is used to amplify the P51 CRISPR backbone using primers CRE1 & CRE4 provided by Andrew. Primers were diluted 1:10 from 100 mM stock
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	25 μL
-!  Reactants
+CRE1 Primer	2.5 μL
-! Volume
+CRE4 Primer	2.5 μL
+P51 DNA	2.0 μL
-|-
+Q5 Polymerase 	0.5 μL
-| Q5 Master Mix
+Nuclease Free H2O	17.5 μL
-| 25 ?L
+Final Reaction Volume	50.0 μL
+_t
-|-
-| CRE1 Primer
-| 2.5 ?L
-|-
-| CRE4 Primer
-| 2.5 ?L
-|-
-| P51 DNA
-| 2.0 ?L
-|-
-| Q5 Polymerase
-| 0.5 ?L
-|-
-| Nuclease Free H2O
-| 17.5 ?L
-|-
-| Final Reaction Volume
-| 50.0 ?L
-|-
-|}
 Thermocycler set as follows for the reaction:
-{| class = "wikitable"
+_t Initial Denature	98°C/30 sec
-|-
+Denature	98°C/20 sec
-!  Initial Denature
+Anneal	60°C/20 sec
-! 98°C/30 sec
+Extend	72°C/3 min
+_t
-|-
-| Denature
-| 98°C/20 sec
-|-
-| Anneal
-| 60°C/20 sec
-|-
-| Extend
-| 72°C/3 min
-|-
-|}
 Restriction Digestion performed on P51 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
-?L sample
+μL sample
-.0	?L DPN1 (100 fold dilution)
+.0	μL DPN1 (100 fold dilution)
-.0 ?L 10X CutSmart Buffer
+.0 μL 10X CutSmart Buffer
 PCR performed to dimerize each of the gRNA primers previously designed (729, 666, 756, SC1, and SC2) following the cPEC protocol and using the following amounts:
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	25.0 μL
-!  Reactants
+Primer	1.7 μL
-! Volume
+Primer 	2.0 μL
+Q5 Polymerase 	0. 5 μL
-|-
+Nuclease Free H2O	19.5 μL
-| Q5 Master Mix
+Final Reaction Volume	50.0 μL
-| 25.0 ?L
+_t
-|-
-| Primer
-| 1.7 ?L
-|-
-| Primer
-| 2.0 ?L
-|-
-| Q5 Polymerase
-| 0. 5 ?L
-|-
-| Nuclease Free H2O
-| 19.5 ?L
-|-
-| Final Reaction Volume
-| 50.0 ?L
-|-
-|}
 Nanodrop performed on some of these products (due to timing) to check for purity and concentration (results below). It is assumed that the other samples have high enough DNA levels to proceed.
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+	405.5	1.78	0.67
-!  Sample
+SC2	393.2	1.80	0.67
-! ng/ ?L
+CasC	455.2	1.5	0.60
-! 260/280
+_t
-! 260/230
-|-
-| 729
-| 405.5
-| 1.78
-| 0.67
-|-
-| SC2
-| 393.2
-| 1.80
-| 0.67
-|-
-| CasC
-| 455.2
-| 1.5
-| 0.60
-|-
-|}
 cPEC performed to combine the P51 amplification and the each of the dimerized gRNAs separately following the cPEC protocol and using the following amounts:
@@ Line 786: / Line 373: @@
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	12.5 μL
-!  Reactants
+Insert (our primer dimers)	1.7 μL
-! Volume
+Template DNA	2.0 μL
+Q5 Polymerase 	0.25 μL
-|-
+Nuclease Free H2O	10.05 μL
-| Q5 Master Mix
+Final Reaction Volume	25.0 μL
-| 12.5 ?L
+_t
-|-
-| Insert (our primer dimers)
-| 1.7 ?L
-|-
-| Template DNA
-| 2.0 ?L
-|-
-| Q5 Polymerase
-| 0.25 ?L
-|-
-| Nuclease Free H2O
-| 10.05 ?L
-|-
-| Final Reaction Volume
-| 25.0 ?L
-|-
-|}
 Repeat of Gibson to combine cas9 and CmR (pSB1C3 backbone) with cas9 PCR product. Gibson protocol was followed for the reaction and included a positive control (same as reaction completed on 5/30/14).
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Gibson Master Mix 	10 μL
-!  Reactants
+Cas9	1.6 μL (50 ng)
-! Volume
+Cmr	2.4 μL (143 ng)
+Nuclease Free H2O	6 μL
-|-
+Final Reaction Volume	20.0 μL
-| Gibson Master Mix
+_t
-| 10 ?L
-|-
-| Cas9
-| 1.6 ?L (50 ng)
-|-
-| Cmr
-| 2.4 ?L (143 ng)
-|-
-| Nuclease Free H2O
-| 6 ?L
-|-
-| Final Reaction Volume
-| 20.0 ?L
-|-
-|}
 Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.
@@ Line 852: / Line 396: @@
 O/N cell cultures made from plates in order to perform a Tet titration tomorrow. 2 tubes of each were made by taking a single colony and mixing with 5 mL of LB and antibiotics as listed below before incubation.
-{| class = "wikitable"
+_t Sample	Antibiotic
-|-
+BWF+ w/ cas	5 μL Kan + 10 μL Amp
-!  Sample
+BW w/ cas	5 μL Kan + 10 μL Amp
-! Antibiotic
+BWF+	5 μL Kan
+BW	5 μL Kan
-|-
+BWF+ (not transformed)	5 μL Kan + 10 μL Tet
-| BWF+ w/ cas
+_t
-| 5 ?L Kan + 10 ?L Amp
-|-
-| BW w/ cas
-| 5 ?L Kan + 10 ?L Amp
-|-
-| BWF+
-| 5 ?L Kan
-|-
-| BW
-| 5 ?L Kan
-|-
-| BWF+ (not transformed)
-| 5 ?L Kan + 10 ?L Tet
-|-
-|}
 /4/14
@@ Line 885: / Line 409: @@
 *Cancelled as cell growth had already neared plateau values
-{| class = "wikitable"
+_t Sample	0 ng/mL	0.5 ng/mL	5 ng/mL	20 ng/mL	50 ng/mL	250 ng/mL	1000 ng/mL	Chloro
-|-
+BW	2.38	2.401	2.389	2.382	2.386	2.398	2.373	2.379
-!  Sample
+BWF+	2.379	2.376	2.428	2.390	2.378	2.365	2.378	2.386
-! 0 ng/mL
+BWcas	2.391	2.356	2.367	2.355	2.366	2.330	2.362	2.382
-! 0.5 ng/mL
+BWF+ cas	2.438	2.447	2.448	2.434	2.436	2.415	2.419	2.415
-! 5 ng/mL
+BWF+ Tet	2.135	2.146	2.141	2.152	2.128	2.127	2.134	2.134
-! 20 ng/mL
+_t
-! 50 ng/mL
-! 250 ng/mL
-! 1000 ng/mL
-! Chloro
-|-
-| BW
-| 2.38
-| 2.401
-| 2.389
-| 2.382
-| 2.386
-| 2.398
-| 2.373
-| 2.379
-|-
-| BWF+
-| 2.379
-| 2.376
-| 2.428
-| 2.390
-| 2.378
-| 2.365
-| 2.378
-| 2.386
-|-
-| BWcas
-| 2.391
-| 2.356
-| 2.367
-| 2.355
-| 2.366
-| 2.330
-| 2.362
-| 2.382
-|-
-| BWF+ cas
-| 2.438
-| 2.447
-| 2.448
-| 2.434
-| 2.436
-| 2.415
-| 2.419
-| 2.415
-|-
-| BWF+ Tet
-| 2.135
-| 2.146
-| 2.141
-| 2.152
-| 2.128
-| 2.127
-| 2.134
-| 2.134
-|-
-|}
 New O/N cell cultures made to do the titration on 6/5/14. Same amounts as shown above were used except only one of each was made.  No cells added to O/N culture on 6/4/14, that is why procedure was repeated.
@@ Line 962: / Line 424: @@
-{| class = "wikitable"
+_t Reactants 	Volume
-|-
+Q5 Master Mix	10.0 μL
-!  Reactants
+Cas9 DNA	5.0 μL
-! Volume
+CmR DNA	5.0 μL
+Nuclease Free H2O	0.00 μL
+Final Reaction Volume	20.0 μL
-|-
+_t
-| Q5 Master Mix
+Thermocycler was varied for each of the three cPEC reactions as listed below:
-| 10.0 ?L
+_t	cPEC1	cPEC2	cPEC3
-|-
-| Cas9 DNA
-| 5.0 ?L
-|-
-| CmR DNA
-| 5.0 ?L
-|-
-| Nuclease Free H2O
-| 0.00 ?L
-|-
-| Final Reaction Volume
-| 20.0 ?L
-|-
-|  _t
-|-
-|
-|-
-| Thermocycler was varied for each of the three cPEC reactions as listed below:
-|-
-|
-|-
-|}
 Initial Denature	98°C/30 sec	98°C/30 sec	98°C/30 sec
 Denature	98°C/10 sec	98°C/20 sec	98°C/10 sec
@@ Line 1,011: / Line 445: @@
 Nanodrop done on the three cPEC casC reactions with results listed below:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+CasC1	489.3	1.80	0.72
-!  Sample
+CasC2	577.7	1.76	0.59
-! ng/ ?L
+CasC3	542.5	1.80	0.74
-! 260/280
+ _t
-! 260/230
+DNA cleanup done on these three casC cPEC reactions, followed by another nanodrop to examine purity with the results below.
-|-
-| CasC1
-| 489.3
-| 1.80
-| 0.72
-|-
+_t Sample	ng/ μL	260/280	260/230
-| CasC2
-| 577.7
-| 1.76
-| 0.59
-|-
-| CasC3
-| 542.5
-| 1.80
-| 0.74
-|-
-|  _t
-|-
-|
-|-
-| DNA cleanup done on these three casC cPEC reactions, followed by another nanodrop to examine purity with the results below.
-|-
-|
-|-
-|
-|-
-|}
 CasC1	13.4	1.99	1.61
 CasC2	11.1	2.02	1.15
 CasC3*	9.9	1.84	1.21
   _t
-% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 122V for ~50 min.
+% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 122V for ~50 min.
-{| class = "wikitable"
+_t Well	Loaded
-|-
+	2-Log DNA Ladder
-!  Well
+	cPEC1
-! Loaded
+	cPEC2
+	cPEC3
+	GA Control
+	GA Positive Control
+	CasC
+	H2O Control
+  _t
+[[File:UCB-induced cc9- gel-140605.jpg]]
-|-
-| 1
-| 2-Log DNA Ladder
-|-
+Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
-| 2
-| cPEC1
+_t 	Time 0	Time 1	Time 2	Time 3	Time 4	Time 5*	Time 6
-|-
-| 3
-| cPEC2
-|-
-| 4
-| cPEC3
-|-
-| 5
-| GA Control
-|-
-| 6
-| GA Positive Control
-|-
-| 7
-| CasC
-|-
-| 8
-| H2O Control
-|-
-|  _t
-|-
-| [[File:UCB-induced cc9- gel-140605.jpg]]
-|-
-|
-|-
-|
-|-
-| Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
-|-
-|
-|-
-|}
 BWF+	0.041	0.047	0.114	0.283	0.597	0.065	0.171
 BWF+ w/cas9	0.035	0.046	0.110	0.262	0.530	0.059	0.154
-{| class = "wikitable"
+_t
-|-
-!
-|-
+*At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.
-|
-|-
+/6/14
-| *At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.
+Plates with the Gibson Assembly products have shown very slow growth. Seven colonies are visible on the Gibson plate (shown with positive control plate below).
+[[File:UCB-induced cc9-plate-01-140606.jpg]]
+[[File:UCB-induced cc9-plate-02-140606.jpg]]
-|-
+Above left: Gibson Assembly plate with 7 colonies. Above Right: Gibson Assembly positive control plate.
-|
-|-
+Week 5
-| 6/6/14
-|-
+/9/14
-|
+Mini-preps were done following manufacturer's protocol except samples were centrifuged 4 minutes at the end (step 9). Ten samples were done that included 7 GA colonies, 1 GA control (labeled GA8), P50, and P51. All were subjected to a nanodrop and a restriction digestion with BamHI and EcoRV was performed on all the samples all except P51.  The nanodrop results are shown below:
-|-
-| Plates with the Gibson Assembly products have shown very slow growth. Seven colonies are visible on the Gibson plate (shown with positive control plate below).
+_t Sample	ng/ μL	260/280	260/230
-|-
-| [[File:UCB-induced cc9-plate-01-140606.jpg]]
-|-
-| [[File:UCB-induced cc9-plate-02-140606.jpg]]
-|-
-|
-|-
-| Above left: Gibson Assembly plate with 7 colonies. Above Right: Gibson Assembly positive control plate.
-|-
-|
-|-
-| Week 5
-|-
-|
-|-
-| 6/9/14
-|-
-|
-|-
-| Mini-preps were done following manufacturer's protocol except samples were centrifuged 4 minutes at the end (step 9). Ten samples were done that included 7 GA colonies, 1 GA control (labeled GA8), P50, and P51. All were subjected to a nanodrop and a restriction digestion with BamHI and EcoRV was performed on all the samples all except P51.  The nanodrop results are shown below:
-|-
-|
-|-
-|
-|-
-|}
 GA1	80.5	1.88	2.04
 GA2	87.8	1.90	2.14
@@ Line 1,188: / Line 509: @@
 P50	91.3	1.88	2.12
 P51	65.8	1.88	2.03
-{| class = "wikitable"
+_t
-|-
-!
-|-
+Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.
-|
-|-
-| Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.
+_t Lane	Sample
-|-
-|
-|-
-|
-|-
-|}
 	2-Log DNA Ladder
 	GA1
@@ Line 1,221: / Line 530: @@
 	Undigested GA3
 	Undigested P50
-{| class = "wikitable"
+_t
-|-
+[[File:UCB-induced cc9- gel- 140609.jpg]]
-!
-|-
-| [[File:UCB-induced cc9- gel- 140609.jpg]]
+Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ½ containing Kan and ½ containing Amp and Kan. The plates were made with increasing amounts of ATC.
-|-
+gRNAs from cPEC were transformed and plated according to protocol with controls.
-|
-|-
-|
+AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:
-|-
+_t Reactants	PCR AmilCP	Original AmilCP
-| Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ? containing Kan and ? containing Amp and Kan. The plates were made with increasing amounts of ATC.
+Buffer 3	1.25 μL	1.25 μL
+BxsI	1.0 μL	2.5 μL
+DNA (125 ng)	4.0 μL	1.25 μL
+Nuclease Free H2O	6.25 μL	7.5 μL
+Final Reaction Volume	12.5 μL	12.5 μL
+_t
+/10/14
+cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.
+First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 μL of each to 40 μL nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 μL of each forward and reverse primer dimer that were added to 40 μL of N.F. H2O to produce a dilution (1:1:8). 5 μL of each primer mix was added to 25 μL of the Q5 master mix and 20 μL N.F. H2O to make a 50 μL total reaction for each sample.Products were checked by gel electrophoresis (shown below).
+[[File:UCB-induced cc9- gel-140610.jpg]]
+Next, a gel purification of a PCR P51 product was performed (to select for linear DNA). This product was digested with DpnI. A PCR cleanup was made of other P51 product that was not gel extracted. All of these steps were done following their recommended protocols.
-|-
+Finally, cPEC reaction was done to combine the gel extracted P51 product with each of the gRNAs using the following amounts. The thermocycler set the same as on 6/3/14:
-|
-|-
-| gRNAs from cPEC were transformed and plated according to protocol with controls.
-|-
+_t  Reactants	Volume
-|
+Q5 Master Mix	25.0 μL
+Primer Mix	2.5 μL
-|-
+DNA (1:10 dilution)	1.0 μL
-|
+Nuclease Free H2O	25.5 μL
+Final Reaction Volume	54.0 μL
-|-
-| AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:
-|-
-|
-|-
-|}
-Buffer 3	1.25 ?L	1.25 ?L
-BxsI	1.0 ?L	2.5 ?L
-DNA (125 ng)	4.0 ?L	1.25 ?L
-Nuclease Free H2O	6.25 ?L	7.5 ?L
-Final Reaction Volume	12.5 ?L	12.5 ?L
-{| class = "wikitable"
-|-
-!
-|-
-| 6/10/14
-|-
-|
-|-
-| cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.
-|-
-|
-|-
-| First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 ?L of each to 40 ?L nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 ?L of each forward and reverse primer dimer that were added to 40 ?L of N.F. H2O to produce a dilution (1:1:8). 5 ?L of each primer mix was added to 25 ?L of the Q5 master mix and 20 ?L N.F. H2O to make a 50 ?L total reaction for each sample.Products were checked by gel electrophoresis (shown below).
-|-
-| [[File:UCB-induced cc9- gel-140610.jpg]]
-|-
-|
-|-
-| Next, a gel purification of a PCR P51 product was performed (to select for linear DNA). This product was digested with DpnI. A PCR cleanup was made of other P51 product that was not gel extracted. All of these steps were done following their recommended protocols.
-|-
-|
-|-
-| Finally, cPEC reaction was done to combine the gel extracted P51 product with each of the gRNAs using the following amounts. The thermocycler set the same as on 6/3/14:
-|-
-|
-|-
-|
-|-
-|}
-Q5 Master Mix	25.0 ?L
-Primer Mix	2.5 ?L
-DNA (1:10 dilution)	1.0 ?L
-Nuclease Free H2O	25.5 ?L
-Final Reaction Volume	54.0 ?L
   _t
 Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+P51	416.9	1.82	0.67
-!  Sample
+AmilCP	430.5	1.82	0.67
-! ng/ ?L
+	472.2	1.80	0.73
-! 260/280
+	479.6	1.78	0.73
-! 260/230
+	475.8	1.80	0.73
+	485.4	1.79	0.75
-|-
+SC1	464.6	1.80	0.73
-| P51
+SC2	475.1	1.80	0.74
-| 416.9
+_t
-| 1.82
-| 0.67
-|-
-| AmilCP
-| 430.5
-| 1.82
-| 0.67
-|-
-| 158
-| 472.2
-| 1.80
-| 0.73
-|-
-| 756
-| 479.6
-| 1.78
-| 0.73
-|-
-| 666
-| 475.8
-| 1.80
-| 0.73
-|-
-| 729
-| 485.4
-| 1.79
-| 0.75
-|-
-| SC1
-| 464.6
-| 1.80
-| 0.73
-|-
-| SC2
-| 475.1
-| 1.80
-| 0.74
-|-
-|}
 Titration Experiment: Done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. The plan is to make 50 mL of BWF+ w/cas9 and BWF+ w/o cas9 with a starting OD600 of 0.05. These will be placed in an incubator and allowed to grow until reaching 0.5. They will then be diluted again to 0.05 (in 50 mL). Serial dilutions will be made and plated on the prepared plates which will be incubated overnight.
@@ Line 1,374: / Line 584: @@
 Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
-{| class = "wikitable"
+_t Sample	OD	Amount added to 50 mL
-|-
+BWF+	0.544	460 μL
-!  Sample
+BWF+ w/cas9	0.556	450 μL
-! OD
+_t
-! Amount added to 50 mL
-|-
-| BWF+
-| 0.544
-| 460 ?L
-|-
-| BWF+ w/cas9
-| 0.556
-| 450 ?L
-|-
-|}
-{| class = "wikitable"
-|-
-!  Time
-! BWF+
-! BWF+ w/cas9
-|-
-| T0
-| 0.059
-| 0.058
-|-
-| T1
-| 0.081
-| 0.078
-|-
-| T2
-| 0.180
-| 0.168
-|-
-| T3
-| 0.291
-| 0.272
-|-
-| T4
-| 0.398
-| 0.366
-|-
-| T5
-| 0.538
-| 0.489
-|-
-| T6*
-| 0.968
-| 0.874
-|-
-| T7
-| 0.165
-| 0.153
-|-
-| T8
-| 0.414
-| 0.396
-|-
-| T9†
-| 0.561
-| 0.490
-|-
-| T10
-| 0.057
-| 0.056
-|-
+_t Time	BWF+	BWF+ w/cas9
-|}
+T0	0.059	0.058
+T1	0.081	0.078
+T2	0.180	0.168
+T3	0.291	0.272
+T4	0.398	0.366
+T5	0.538	0.489
+T6*	0.968	0.874
+T7	0.165	0.153
+T8	0.414	0.396
+T9†	0.561	0.490
+T10	0.057	0.056
+_t
 *At T6, the cultures were again diluted down (5.17 mL BWF+, 5.72 mL BWF+ w/cas9 into 50 mL).
 †After T9, 4.898 mL BWF+ and 4.278 mL BWF+ w/ cas9 were again diluted into 50 mL LB. A final OD was taken to ensure that the final OD was close to 0.05 before the cells were plated.
@@ Line 1,467: / Line 613: @@
 As seen from the images in the table below, the cas9 protein inhibits growth of the cells even prior to removal of repression by the TetR repressor, demonstrating some leaky expression. Increasing ATC concentrations results in increased cas9 expression in the cells as expected.
 [[File:UCB-induced cc9-titration plates-140611.JPG]]
@@ Line 1,473: / Line 620: @@
 Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+GE P51	13.7	1.94	1.18
-!  Sample
+GE AmilCP PCR	27.3	1.81	1.19
-! ng/ ?L
+GE cas9 PCR	7.8	1.98	0.62
-! 260/280
+AmilCP DpnI digest	644.8	1.6	0.65
-! 260/230
+P50 PCR	493.9	1.8	0.73
+GA1 MP	107.9	1.87	1.99
-|-
+GA2 MP	144.9	1.88	2.11
-| GE P51
+GA3 MP	168.3	1.92	2.29
-| 13.7
+GA4 MP	131.1	1.91	1.93
-| 1.94
+GA5 MP	310.6	1.88	2.34
-| 1.18
+-1	125.3	1.88	2.11
+-2	178.3	1.90	2.17
-|-
+-3	172.6	1.84	1.81
-| GE AmilCP PCR
+-1	420.7	1.87	2.25
-| 27.3
+-2	106.8	1.89	1.99
-| 1.81
+  _t
-| 1.19
+/12/14
-|-
-| GE cas9 PCR
+Transformations done of all 6 gRNA cPEC reactions, P50 (+control), and P51 PCR (-control)
-| 7.8
+Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).
-| 1.98
-| 0.62
+_t Sample	ng/ μL	260/280	260/230
-|-
-| AmilCP DpnI digest
-| 644.8
-| 1.6
-| 0.65
-|-
-| P50 PCR
-| 493.9
-| 1.8
-| 0.73
-|-
-| GA1 MP
-| 107.9
-| 1.87
-| 1.99
-|-
-| GA2 MP
-| 144.9
-| 1.88
-| 2.11
-|-
-| GA3 MP
-| 168.3
-| 1.92
-| 2.29
-|-
-| GA4 MP
-| 131.1
-| 1.91
-| 1.93
-|-
-| GA5 MP
-| 310.6
-| 1.88
-| 2.34
-|-
-| 729-1
-| 125.3
-| 1.88
-| 2.11
-|-
-| 729-2
-| 178.3
-| 1.90
-| 2.17
-|-
-| 729-3
-| 172.6
-| 1.84
-| 1.81
-|-
-| 756-1
-| 420.7
-| 1.87
-| 2.25
-|-
-| 756-2
-| 106.8
-| 1.89
-| 1.99
-|-
-|  _t
-|-
-|
-|-
-| 6/12/14
-|-
-|
-|-
-| Transformations done of all 6 gRNA cPEC reactions, P50 (+control), and P51 PCR (-control)
-|-
-| Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).
-|-
-|
-|
-|-
-|}
 Cas9	5.5	2.12	0.59
   _t
 P50 PCR amplification performed using the following amounts:
--	25 ?L Master Mix
+-	25 μL Master Mix
--	2.5 ?L of forward primer (10 ?M)
+-	2.5 μL of forward primer (10 μM)
--	2.5 ?L of reverse primer (10 ?M)
+-	2.5 μL of reverse primer (10 μM)
--	1.0 ?L DNA (1:50 dilution)
+-	1.0 μL DNA (1:50 dilution)
--	19.0 ?L NF H2O
+-	19.0 μL NF H2O
 Two samples prepared with different thermocycler settings as follows:
-{| class = "wikitable"
+_t 	1st Round	2nd Round
-|-
+Initial Denature	98°C/30 sec	98°C/2 min
-!
+Denature	98°C/30 sec	98°C/30 sec
-! 1st Round
+Anneal	69°C/30 sec	68°C/30 sec
-! 2nd Round
+Extend	72°C/3.5 min	72°C/5.5 min
+# of cycles	34	30
-|-
+Final Extension	72°C/2 min	72°C/5 min
-| Initial Denature
+_t
-| 98°C/30 sec
-| 98°C/2 min
-|-
-| Denature
-| 98°C/30 sec
-| 98°C/30 sec
-|-
-| Anneal
-| 69°C/30 sec
-| 68°C/30 sec
-|-
-| Extend
-| 72°C/3.5 min
-| 72°C/5.5 min
-|-
-| # of cycles
-| 34
-| 30
-|-
-| Final Extension
-| 72°C/2 min
-| 72°C/5 min
-|-
-|}
 Overnight cultures prepared on GA1-GA5, 729-1, 729-2, 729-3, and 759-1, 759-2
-Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 ?L Amp (50 ng/mL)
+Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 μL Amp (50 ng/mL)
 /13/14
@@ Line 1,651: / Line 671: @@
 Mini-prep done on O/N cultures SC1-1 and SC1-2 followed by a nanodrop. Remainder of culture was spun down into a pellet and frozen.
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+SC1-1	30.7	2.02	2.06
-!  Sample
+SC1-2	32.5	1.94	1.48
-! ng/ ?L
+  _t
-! 260/280
-! 260/230
+Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.
-|-
+_t Sample	Buffer 3	10X BSA	Enzyme 1	Enzyme 2	Enzyme Mix*	DNA (200 ng)	MQ H2O	Total Volume
-| SC1-1
+-1	2.0 μL	2.0 μL	0.5 μL EagI	0.5 μL XbaI	--	1.0 μL	14.0 μL	20.0 μL
-| 30.7
+-2	2.0 μL	2.0 μL	0.5 μL EagI	0.5 μL XbaI	--	1.87 μL	13.13 μL	20.0 μL
-| 2.02
+GA1	2.0 μL	2.0 μL	--	--	1.0 μL	1.85 μL	13.15 μL	20.0 μL
-| 2.06
+GA2	2.0 μL	2.0 μL	--	--	1.0 μL	1.38 μL	13.62 μL	20.0 μL
+GA3	2.0 μL	2.0 μL	--	--	1.0 μL	1.19 μL	13.81 μL	20.0 μL
-|-
+GA4	2.0 μL	2.0 μL	--	--	1.0 μL	1.53 μL	13.47 μL	20.0 μL
-| SC1-2
+GA5	2.0 μL	2.0 μL	--	--	1.0 μL	0.64 μL	14.36 μL	20.0 μL
-| 32.5
+P51	2.0 μL	2.0 μL	--	--	1.0 μL	4.0 μL	11.0 μL	20.0 μL
-| 1.94
+Cas9	2.0 μL	2.0 μL	--	--	1.0 μL	2.0 μL	13.0 μL	20.0 μL
-| 1.48
+_t
+*Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI
-|-
-|  _t
+Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
+_t Lane	Sample
-|-
-|
-|-
-| Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.
-|-
-|
-|-
-|}
--1	2.0 ?L	2.0 ?L	0.5 ?L EagI	0.5 ?L XbaI	--	1.0 ?L	14.0 ?L	20.0 ?L
--2	2.0 ?L	2.0 ?L	0.5 ?L EagI	0.5 ?L XbaI	--	1.87 ?L	13.13 ?L	20.0 ?L
-GA1	2.0 ?L	2.0 ?L	--	--	1.0 ?L	1.85 ?L	13.15 ?L	20.0 ?L
-GA2	2.0 ?L	2.0 ?L	--	--	1.0 ?L	1.38 ?L	13.62 ?L	20.0 ?L
-GA3	2.0 ?L	2.0 ?L	--	--	1.0 ?L	1.19 ?L	13.81 ?L	20.0 ?L
-GA4	2.0 ?L	2.0 ?L	--	--	1.0 ?L	1.53 ?L	13.47 ?L	20.0 ?L
-GA5	2.0 ?L	2.0 ?L	--	--	1.0 ?L	0.64 ?L	14.36 ?L	20.0 ?L
-P51	2.0 ?L	2.0 ?L	--	--	1.0 ?L	4.0 ?L	11.0 ?L	20.0 ?L
-Cas9	2.0 ?L	2.0 ?L	--	--	1.0 ?L	2.0 ?L	13.0 ?L	20.0 ?L
-{| class = "wikitable"
-|-
-!
-|-
-| *Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI
-|-
-|
-|-
-| Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-|-
-|}
 	2-Log DNA Ladder
 	756-1
@@ Line 1,719: / Line 704: @@
 	GA5
 	Cas9 Digest
-{| class = "wikitable"
+_t
-|-
+ [[File:UCB-induced cc9-gel-140613.JPG]]
-!
-|-
-|  [[File:UCB-induced cc9-gel-140613.JPG]]
-|-
+From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 μL sample plus 1 μL loading dye for each) as shown below:
-|
+_t Lane	Sample
-|-
-|
-|-
-| From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 ?L sample plus 1 ?L loading dye for each) as shown below:
-|-
-|}
 	2-Log DNA Ladder
 	GA4
@@ Line 1,745: / Line 718: @@
 	Cas9 GE
 	GA4 MP
-{| class = "wikitable"
+_t
-|-
+[[File:UCB-induced cc9-gel-02-140613.JPG]]
-!
-|-
-| [[File:UCB-induced cc9-gel-02-140613.JPG]]
-|-
+/14/14
-|
+Gibson Assembly performed both with and without 2% DMSO. Each 20 μL reaction contained 10 μL Gibson Master Mix with the following as listed:
+Reaction #1
+-	6.5 μL GE Cas9
+-	3.5 μL GE AmilCP
+Reaction #2
+-	1 μL P50 PCR Product (1:10)
+-	5 μL GE AmilCP
+-	4 μL MQ H2O
+Reaction #3
+-	1 μL P50 PCR Product (1:10)
+-	5 μL GE AmilCP
+-	4 μL 1:10 dilution of 2% DMSO
+Bacterial Transformation: Done according to protocol
+-	GA1-GA3 diluted 1:4 transformed with 2 μL (100 μL each)
+-	BWF+ w/Cas9 from S.B. (25 μL full strength and 25 μL 1:10 dilution)
+-	BWF+ (25 μL full strength and 25 μL 1:10 dilution)
+-	NEB competent cells with P51 (50 μL)
+-	Competent cells with P51 (50 μL) for comparison
+Week 6
-|-
+/15/14
-|
+GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ μL Amp.
-|-
+/16/14
-| 6/14/14
+Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:
-|-
-|
+_t Sample	ng/ μL	260/280	260/230
-|-
-|
-|-
-| Gibson Assembly performed both with and without 2% DMSO. Each 20 ?L reaction contained 10 ?L Gibson Master Mix with the following as listed:
-|-
-| Reaction #1
-|
-|-
-| -
-| 6.5 ?L GE Cas9
-|-
-| -
-| 3.5 ?L GE AmilCP
-|-
-|
-|-
-| Reaction #2
-|-
-| -
-| 1 ?L P50 PCR Product (1:10)
-|-
-| -
-| 5 ?L GE AmilCP
-|-
-| -
-| 4 ?L MQ H2O
-|-
-|
-|-
-| Reaction #3
-|-
-| -
-| 1 ?L P50 PCR Product (1:10)
-|-
-| -
-| 5 ?L GE AmilCP
-|-
-| -
-| 4 ?L 1:10 dilution of 2% DMSO
-|-
-| Bacterial Transformation: Done according to protocol
-|-
-| -
-| GA1-GA3 diluted 1:4 transformed with 2 ?L (100 ?L each)
-|-
-| -
-| BWF+ w/Cas9 from S.B. (25 ?L full strength and 25 ?L 1:10 dilution)
-|-
-| -
-| BWF+ (25 ?L full strength and 25 ?L 1:10 dilution)
-|-
-| -
-| NEB competent cells with P51 (50 ?L)
-|-
-| -
-| Competent cells with P51 (50 ?L) for comparison
-|-
-|
-|-
-| Week 6
-|-
-|
-|-
-| 6/15/14
-|-
-|
-|-
-| GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ ?L Amp.
-|-
-|
-|-
-|
-|-
-| 6/16/14
-|-
-|
-|-
-| Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:
-|-
-|
-|-
-|}
 -1	17.5	2.08	1.94
 -2	54.1	1.93	1.96
@@ Line 1,892: / Line 771: @@
   _t
-Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 ?L and MQ H2O was not added).
+Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 μL and MQ H2O was not added).
--	1.0 ?L Buffer D
+-	1.0 μL Buffer D
--	3.0 ?L Enzyme
+-	3.0 μL Enzyme
--	3.0 ?L sample DNA
+-	3.0 μL sample DNA
--	3.0 ?L MQ H2O
+-	3.0 μL MQ H2O
--	Total Reaction Volume = 10 ?L
+-	Total Reaction Volume = 10 μL
 Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-{| class = "wikitable"
+_t Lane	Sample
-|-
+	2-Log DNA Ladder
-!  Lane
+	1-1
-! Sample
+	1-2
+	1-3
-|-
+	2-1
-| 1
+	2-2
-| 2-Log DNA Ladder
+	2-3
+	2-4
-|-
+	2-5
-| 2
+	2-6
-| 1-1
+	2-7
+	2-8
-|-
+_t
-| 3
-| 1-2
-|-
-| 4
-| 1-3
-|-
-| 5
-| 2-1
-|-
-| 6
-| 2-2
-|-
-| 7
-| 2-3
-|-
-| 8
-| 2-4
-|-
-| 9
-| 2-5
-|-
-| 10
-| 2-6
-|-
-| 11
-| 2-7
-|-
-| 12
-| 2-8
-|-
-|}
 Repeated digestion (protocol above) on select samples to run again for more definition on gel. Samples are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-{| class = "wikitable"
+_t Lane	Sample
-|-
+	2-Log DNA Ladder
-!  Lane
+	1-1
-! Sample
+	1-2
+	2-1
-|-
+	sb1c3
-| 1
+	Digested sb1c3
-| 2-Log DNA Ladder
+	Digested p50
+	2-Log DNA Ladder
-|-
+_t
-| 2
-| 1-1
-|-
-| 3
-| 1-2
-|-
-| 4
-| 2-1
-|-
-| 5
-| sb1c3
-|-
-| 6
-| Digested sb1c3
-|-
-| 7
-| Digested p50
-|-
-| 8
-| 2-Log DNA Ladder
-|-
-|}
 [[File:UCB-induced cc9-gel-140616.JPG]]
 Bacterial Transformation: Done according to protocol
--	7 CPEC products including p51 as the negative control (10 ?L  1:4 dilution) with 40 ?L  DH5?
+-	7 CPEC products including p51 as the negative control (10 μL  1:4 dilution) with 40 μL  DH5α
--	BWF+ w (40?L)/ S.B. cas9 (1 ?L)
+-	BWF+ w (40μL)/ S.B. cas9 (1 μL)
--	BWF+ (1 ?L)
+-	BWF+ (1 μL)
 Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen
@@ Line 2,010: / Line 819: @@
 /17/14
-{| class = "wikitable"
+_t Sample	Buffer 	Enzyme 1	EnzymeMix*	DNA (200 ng)	MQ H2O	Total Volume
-|-
+GA x 1-1	1.0 μL cutsmart	3.0 μL XbaI (1:50)	--	3.0 μL	3.0 μL	10.0 μL
-!  Sample
+GA x 1-2	1.0 μL cutsmart	3.0 μL XbaI (1:50)	--	    3.0  μL	3.0 μL	10.0 μL
-! Buffer
+GA x 2-1	1.0 μL cutsmart	3.0 μL XbaI (1:50)	--	3.0 μL	3.0 μL	10.0 μL
-! Enzyme 1
+p50 linear	1.0 μL cutsmart	3.0 μL SepI (1:50)	--	3.0 μL	3.0 μL	10.0 μL
-! EnzymeMix*
+GA H 1-1	1.0 μL Fast green digest	--	3.0 μL	3.0 μL	3.0 μL	10.0 μL
-! DNA (200 ng)
+GA H 1-2	1.0 μL Fast green digest	--	3.0 μL	3.0 μL	3.0 μL	10.0 μL
-! MQ H2O
+GA H 2-1	1.0 μL Fast green digest	--	3.0 μL	3.0 μL	3.0 μL	10.0 μL
-! Total Volume
+p50	1.0  μL Fast green digest	--	3.0 μL	3.0 μL	3.0 μL	10.0 μL
+_t
-|-
-| GA x 1-1
-| 1.0 ?L cutsmart
-| 3.0 ?L XbaI (1:50)
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| GA x 1-2
-| 1.0 ?L cutsmart
-| 3.0 ?L XbaI (1:50)
-| --
-|     3.0  ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| GA x 2-1
-| 1.0 ?L cutsmart
-| 3.0 ?L XbaI (1:50)
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| p50 linear
-| 1.0 ?L cutsmart
-| 3.0 ?L SepI (1:50)
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| GA H 1-1
-| 1.0 ?L Fast green digest
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| GA H 1-2
-| 1.0 ?L Fast green digest
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| GA H 2-1
-| 1.0 ?L Fast green digest
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-| p50
-| 1.0  ?L Fast green digest
-| --
-| 3.0 ?L
-| 3.0 ?L
-| 3.0 ?L
-| 10.0 ?L
-|-
-|}
 *Enzyme mix: 1:1:23 dilution of xhoI and HindIII
 Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-{| class = "wikitable"
+_t Lane	Sample
-|-
+	2-Log DNA Ladder
-!  Lane
+	GA x 1-1
-! Sample
+	GA x 1-2
+	GA x 2-1
-|-
+	p50
-| 1
+	sb1c3
-| 2-Log DNA Ladder
+	GA H 1-1
+	GA H 1-2
-|-
+	GA H 2-1
-| 2
+	p50
-| GA x 1-1
+	2-Log DNA Ladder
-|-
-| 3
-| GA x 1-2
-|-
-| 4
-| GA x 2-1
-|-
-| 5
-| p50
-|-
-| 6
-| sb1c3
-|-
-| 7
-| GA H 1-1
-|-
-| 8
-| GA H 1-2
-|-
-| 9
-| GA H 2-1
-|-
-| 10
-| p50
-|-
-| 11
-| 2-Log DNA Ladder
-|-
-|
-|-
+_t
-|}
   [[File:UCB-induced cc9-gel-140617.JPG]]
 /18/14
-Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips  were then ejected into  3ml LB+AMP  for O/N culture.The following was added to each tube to make a 25 ?L reaction.
+Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips  were then ejected into  3ml LB+AMP  for O/N culture.The following was added to each tube to make a 25 μL reaction.
--	12.5 ?L 2X Master Mix
+-	12.5 μL 2X Master Mix
--	1.25 ?L of forward primer (10 ?M)
+-	1.25 μL of forward primer (10 μM)
--	1.25 ?L of reverse primer (10 ?M)
+-	1.25 μL of reverse primer (10 μM)
--	10.0 ?L MQ H2O
+-	10.0 μL MQ H2O
 For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).
-{| class = "wikitable"
+_t Initial Denature	98°C/10 min
-|-
+Denature	98°C/30 sec
-!  Initial Denature
+Anneal	65°C/30 sec
-! 98°C/10 min
+Extend	72°C/30 sec
+# of cycles	35
-|-
+Final Extension	72°C/5 min
-| Denature
+_t
-| 98°C/30 sec
-|-
-| Anneal
-| 65°C/30 sec
-|-
-| Extend
-| 72°C/30 sec
-|-
-| # of cycles
-| 35
-|-
-| Final Extension
-| 72°C/5 min
-|-
-|}
 /19/14
 Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.
@@ Line 2,202: / Line 877: @@
 Nanodrops were taken to assess the concentration and purity of the samples:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+H	65.2	1.88	2.21
-!  Sample
+E	56.3	1.91	2.01
-! ng/ ?L
+E	38.9	1.93	2.22
-! 260/280
+B	54.1	1.86	1.54
-! 260/230
+B	69.4	1.88	2.12
+B	66.6	1.82	1.56
+B	69.7	1.83	1.75
+B	61.1	1.89	1.81
+C	52.2	1.86	1.65
+B	43.9	1.86	1.33
+B	57.5	1.83	1.53
+_t
-|-
+Sequencing 12 CPEC products: 12.5 μL DNA + 2.5 μL  10μm primer (CRE22)
-| 2H
-| 65.2
-| 1.88
-| 2.21
-|-
-|      3E
-| 56.3
-| 1.91
-| 2.01
-|-
-| 4E
-| 38.9
-| 1.93
-| 2.22
-|-
-| 5B
-| 54.1
-| 1.86
-| 1.54
-|-
-| 6B
-| 69.4
-| 1.88
-| 2.12
-|-
-| 7B
-| 66.6
-| 1.82
-| 1.56
-|-
-| 8B
-| 69.7
-| 1.83
-| 1.75
-|-
-| 9B
-| 61.1
-| 1.89
-| 1.81
-|-
-| 10C
-| 52.2
-| 1.86
-| 1.65
-|-
-| 11B
-| 43.9
-| 1.86
-| 1.33
-|-
-| 12B
-| 57.5
-| 1.83
-| 1.53
-|-
-|}
-Sequencing 12 CPEC products: 12.5 ?L DNA + 2.5 ?L  10?m primer (CRE22)
-{| class = "wikitable"
+_t	CPEC product
-|-
+	1A
-!
+	1C
-! CPEC product
+	2B
+	2C
-|-
+	3B
-| 1
+	3H
-| 1A
+	4C
+	4D
-|-
+	5E
-| 2
+	5F
-| 1C
+	6C
+	6F
-|-
+_t
-| 3
-| 2B
-|-
-| 4
-| 2C
-|-
-| 5
-| 3B
-|-
-| 6
-| 3H
-|-
-| 7
-| 4C
-|-
-| 8
-| 4D
-|-
-| 9
-| 5E
-|-
-| 10
-| 5F
-|-
-| 11
-| 6C
-|-
-| 12
-| 6F
-|-
-|}
 /26/14
-Sequencing: Total volume 0f 15 ?L
+Sequencing: Total volume 0f 15 μL
-{| class = "wikitable"
+_t	CPEC Product	DNA μL	Primer CRE22	MQ H2O
-|-
+	2B	12.5μL	2.5 μL	--
-!
+	2C	12.0μL	2.5μL	0.5μL
-! CPEC Product
+	3B	10.3μL	2.5μL	2.2μL
-! DNA ?L
+	3H	11μL	2.5μL	1.5μL
-! Primer CRE22
+	4E	12.5μL	2.5μL	--
-! MQ H2O
+	5E	11.3μL	2.5μL	1.2μL
+	11B	12.5μL	2.5μL	--
-|-
+	2H	8μL 	2.5μL	4.5μL
-| 1
+	6B	8μL	2.5μL	4.5μL
-| 2B
+	7B	8μL	2.5μL	4.5μL
-| 12.5?L
+	8B	8μL	2.5μL	4.5μL
-| 2.5 ?L
+	9B	8μL	2.5μL	4.5μL
-| --
+	4C	8μL	2.5μL	4.5μL
+	3E	10μL	2.5μL	2.5μL
-|-
+	5B	10μL	2.5μL	2.5μL
-| 2
+	10C	10μL	2.5μL	2.5μL
-| 2C
+	12B	10μL	2.5μL	2.5μL
-| 12.0?L
-| 2.5?L
-| 0.5?L
-|-
-| 3
-| 3B
-| 10.3?L
-| 2.5?L
-| 2.2?L
-|-
-| 4
-| 3H
-| 11?L
-| 2.5?L
-| 1.5?L
-|-
-| 5
-| 4E
-| 12.5?L
-| 2.5?L
-| --
-|-
-| 6
-| 5E
-| 11.3?L
-| 2.5?L
-| 1.2?L
-|-
-| 7
-| 11B
-| 12.5?L
-| 2.5?L
-| --
-|-
-| 8
-| 2H
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 9
-| 6B
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 10
-| 7B
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 11
-| 8B
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 12
-| 9B
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 13
-| 4C
-| 8?L
-| 2.5?L
-| 4.5?L
-|-
-| 14
-| 3E
-| 10?L
-| 2.5?L
-| 2.5?L
-|-
-| 15
-| 5B
-| 10?L
-| 2.5?L
-| 2.5?L
-|-
-| 16
-| 10C
-| 10?L
-| 2.5?L
-| 2.5?L
-|-
-| 17
-| 12B
-| 10?L
-| 2.5?L
-| 2.5?L
-|-
-|
-|-
+_t
-|}
 Bacterial Transformation of SBcas9• ER: Done according  to protocol.
@@ Line 2,477: / Line 937: @@
 Mini prep done to protocol on CPEC products.
 Nanodrops were taken to assess the concentration and purity of the samples:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+A	66.9	1.90	2.26
-!  Sample
+C	61.3	1.92	2.20
-! ng/ ?L
+B	57.8	1.91	2.27
-! 260/280
+B	69.4	1.88	1.14
-! 260/230
+H	51.7	1.89	2.24
+B	33.6	1.99	1.62
+C	49.8	1.93	2.09
+B	40.5	1.94	2.01
+B	51.9	1.89	1.17
+_t
-|-
+Sequenced all of the above  products (total volume of 15 μL: 10 μL DNA+ 2.5 μLprimer (CRE22)+2 .5 μL  MQ H2O
-| 1A
-| 66.9
-| 1.90
-| 2.26
-|-
-|      1C
-| 61.3
-| 1.92
-| 2.20
-|-
-| 2B
-| 57.8
-| 1.91
-| 2.27
-|-
-| 3B
-| 69.4
-| 1.88
-| 1.14
-|-
-| 3H
-| 51.7
-| 1.89
-| 2.24
-|-
-| 9B
-| 33.6
-| 1.99
-| 1.62
-|-
-| 10C
-| 49.8
-| 1.93
-| 2.09
-|-
-| 11B
-| 40.5
-| 1.94
-| 2.01
-|-
-| 12B
-| 51.9
-| 1.89
-| 1.17
-|-
-|}
-Sequenced all of the above  products (total volume of 15 ?L: 10 ?L DNA+ 2.5 ?Lprimer (CRE22)+2 .5 ?L  MQ H2O
 Exceptions:
--9B received 12.5 ?L DNA + 2.5 ?L primer (CRE22)
+-9B received 12.5 μL DNA + 2.5 μL primer (CRE22)
 -12B received p51 seq1 as the primer
-Glycerol stock of all the above samples  to protocol: 1:1 w/ 40% glycerol for a total volume of 500 ?L each.
+Glycerol stock of all the above samples  to protocol: 1:1 w/ 40% glycerol for a total volume of 500 μL each.
 Positive sequencing results: 1A, 1C, 2B, 3B, 3H, 9B, 10C, 11B, 12B
@@ Line 2,556: / Line 964: @@
 Mini preps done to protocol on the below samples
 Nanodrops were taken to assess the concentration and purity of the samples:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+A	78.7	1.90	2.08
-!  Sample
+C	54.3	1.88	1.76
-! ng/ ?L
+B	335.4	1.48	0.91
-! 260/280
+B	52.1	1.91	1.96
-! 260/230
+H	59.7	1.89	2.03
+_t
-|-
-| 1A
-| 78.7
-| 1.90
-| 2.08
-|-
-|      1C
-| 54.3
-| 1.88
-| 1.76
-|-
-| 2B
-| 335.4
-| 1.48
-| 0.91
-|-
-| 3B
-| 52.1
-| 1.91
-| 1.96
-|-
-| 3H
-| 59.7
-| 1.89
-| 2.03
-|-
-|}
 Week 8
@@ Line 2,604: / Line 980: @@
 Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction.
-{| class = "wikitable"
+_t Sample	ng/ μL
-|-
+p50 Amplicon	27.3
-!  Sample
+      GE AmilCP	493.9
-! ng/ ?L
+_t
-|-
-| p50 Amplicon
-| 27.3
-|-
-|      GE AmilCP
-| 493.9
-|-
-|}
 *Gel extracted
 cPEC performed to combine the p50 amplicon with AmilCP following the cPEC protocol and using the following amounts:
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	12.5 μL
-!  Reactants
+Insert (AmilCP)	5 μL
-! Volume
+Template DNA p50	2.0 μL (1:20 dilution)
+% DMSO	2.0 μL
-|-
+Nuclease Free H2O	3.5 μL
-| Q5 Master Mix
+Final Reaction Volume	25.0 μL
-| 12.5 ?L
+_t
-|-
-| Insert (AmilCP)
-| 5 ?L
-|-
-| Template DNA p50
-| 2.0 ?L (1:20 dilution)
-|-
-| 25% DMSO
-| 2.0 ?L
-|-
-| Nuclease Free H2O
-| 3.5 ?L
-|-
-| Final Reaction Volume
-| 25.0 ?L
-|-
-|}
 Thermocycler conditions:
-{| class = "wikitable"
+_t 	cPEC
-|-
+Initial Denature	98°C/30 sec
-!
+Denature	98°C/10 sec
-! cPEC
+Anneal	60°C/30 sec
+Extend	72°C/105 sec
-|-
+# of cycles	10
-| Initial Denature
+Final Extension	72°C/10 min
-| 98°C/30 sec
+_t
-|-
-| Denature
-| 98°C/10 sec
-|-
-| Anneal
-| 60°C/30 sec
-|-
-| Extend
-| 72°C/105 sec
-|-
-| # of cycles
-| 10
-|-
-| Final Extension
-| 72°C/10 min
-|-
-|}
 Successful Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51 (from 6/30) . O/N cultures of each were then made.
@@ Line 2,703: / Line 1,022: @@
 	*PCR tube lids opened in thermocycler for samples: 8, 12, and positive control
 Colony PCR products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-{| class = "wikitable"
+_t Lane	Sample
-|-
+	2-Log DNA Ladder
-!  Lane
+	1
-! Sample
+	2
+	3
-|-
+	4
-| 1
+	5
-| 2-Log DNA Ladder
+	6
+	7
-|-
+	8
-| 2
+	9
-| 1
+	10
+	11
-|-
+	12
-| 3
+	(+) control
-| 2
+	(-) control
+_t
-|-
-| 4
-| 3
-|-
-| 5
-| 4
-|-
-| 6
-| 5
-|-
-| 7
-| 6
-|-
-| 8
-| 7
-|-
-| 9
-| 8
-|-
-| 10
-| 9
-|-
-| 11
-| 10
-|-
-| 12
-| 11
-|-
-| 13
-| 12
-|-
-| 14
-| (+) control
-|-
-| 15
-| (-) control
-|-
-|}
 /9/14
@@ Line 2,775: / Line 1,044: @@
 Followed by nanodrop with results listed below:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+	89	1.90	2.22
-!  Sample
+	110.9	1.88	2.22
-! ng/ ?L
+	134.4	1.85	1.77
-! 260/280
+	140.9	1.87	2.10
-! 260/230
+	93.6	1.89	2.21
+p50	191.7	1.87	2.35
-|-
+_t
-| 1
-| 89
-| 1.90
-| 2.22
-|-
-| 7
-| 110.9
-| 1.88
-| 2.22
-|-
-| 8
-| 134.4
-| 1.85
-| 1.77
-|-
-| 10
-| 140.9
-| 1.87
-| 2.10
-|-
-| 11
-| 93.6
-| 1.89
-| 2.21
-|-
-| p50
-| 191.7
-| 1.87
-| 2.35
-|-
-|}
 The above samples (except 8) were sent for sequencing.
-Total volume 0f 15 ?L
+Total volume 0f 15 μL
-{| class = "wikitable"
+_t	 Product	DNA μL	Primer CRE22	MQ H2O
-|-
+	1	6.0μL	5.0 μL	4.0μL
-!
+	7	5.0μL	5.0μL	5.0μL
-!  Product
+	10	4.0μL	5.0μL	6.0μL
-! DNA ?L
+	11	6.0μL	5.0μL	4.0μL
-! Primer CRE22
+_t
-! MQ H2O
-|-
-| 1
-| 1
-| 6.0?L
-| 5.0 ?L
-| 4.0?L
-|-
-| 2
-| 7
-| 5.0?L
-| 5.0?L
-| 5.0?L
-|-
-| 3
-| 10
-| 4.0?L
-| 5.0?L
-| 6.0?L
-|-
-| 4
-| 11
-| 6.0?L
-| 5.0?L
-| 4.0?L
-|-
-|}
 Transformation done to protocol on all gRNAs into BWF+
@@ Line 2,867: / Line 1,067: @@
 Site directed Mutagenesis (SDM) performed on original p50, 1, 7, 10, 11 to remove Xba1 from p50
 Primer names: p50TetRXba1F and  p50TetRXba1R
-Primer Mix: 1 ?L forward + 1 ?L reverse + 18 ?L MQ H2O
+Primer Mix: 1 μL forward + 1 μL reverse + 18 μL MQ H2O
 PCR run on the following samples of p50: p50 DNA dilution for original, 1,7,10, and 11
@@ Line 2,878: / Line 1,078: @@
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	12.5 μL
-!  Reactants
+μM primer mix	2.5 μL
-! Volume
+DNA p50	2.0 μL
+Nuclease Free H2O	8.0 μL
-|-
+Final Reaction Volume	25.0 μL
-| Q5 Master Mix
+_t
-| 12.5 ?L
-|-
-| 10 ?M primer mix
-| 2.5 ?L
-|-
-| DNA p50
-| 2.0 ?L
-|-
-| Nuclease Free H2O
-| 8.0 ?L
-|-
-| Final Reaction Volume
-| 25.0 ?L
-|-
-|}
 Thermocycler conditions:
-{| class = "wikitable"
+_t
-|-
+Initial Denature 	98°C/30 sec
-!
+Denature	98°C/30 sec
-!
+Anneal	55°C/30 sec
+Extend	72°C/3.5 minutes
-|-
+# of cycles 	15 cycles
-| Initial Denature
+Final Extension	72°C/5 minutes
-| 98°C/30 sec
+_t
-|-
-| Denature
-| 98°C/30 sec
-|-
-| Anneal
-| 55°C/30 sec
-|-
-| Extend
-| 72°C/3.5 minutes
-|-
-| # of cycles
-| 15 cycles
-|-
-| Final Extension
-| 72°C/5 minutes
-|-
-|}
 SDM performed on 6 gRNAs and the original p51 to remove spe1 site
 			PCR preformed:
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	12.5 μL
-!  Reactants
+μM primer mix	2.5 μL
-! Volume
+DNA p51	2.0 μL
+Nuclease Free H2O	8.0 μL
-|-
+Final Reaction Volume	25.0 μL
-| Q5 Master Mix
+_t
-| 12.5 ?L
+*primer mix: 1 μL forward primer+ 1 μL reverse primer +18 μL MQH2O. Primers varied for each gRNA
-|-
-| 10 ?M primer mix
-| 2.5 ?L
-|-
-| DNA p51
-| 2.0 ?L
-|-
-| Nuclease Free H2O
-| 8.0 ?L
-|-
-| Final Reaction Volume
-| 25.0 ?L
-|-
-|}
-*primer mix: 1 ?L forward primer+ 1 ?L reverse primer +18 ?L MQH2O. Primers varied for each gRNA
 *control consisted of DNA only, no primers
 Thermocycler conditions:
-{| class = "wikitable"
+_t
-|-
+Initial Denature 98	95°C/30 sec
-!
+Denature 98	95°C/30 sec
-!
+Anneal 55	55°C/1min
+Extend 72	68°C/3min
-|-
+# of cycles 	15 cycles
-| Initial Denature 98
+Final Extension 72	3 min
-| 95°C/30 sec
+_t
-|-
-| Denature 98
-| 95°C/30 sec
-|-
-| Anneal 55
-| 55°C/1min
-|-
-| Extend 72
-| 68°C/3min
-|-
-| # of cycles
-| 15 cycles
-|-
-| Final Extension 72
-| 3 min
-|-
-|}
 /11/2014
 All PCR gRNAs DPN1 digested:
-. Add 1 ?l of the Dpn I restriction enzyme (10 U/?l) directly to each
+. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each
 amplification reaction below the mineral oil overlay.
 . Gently and thoroughly mix each reaction mixture by pipetting the
@@ Line 3,016: / Line 1,130: @@
 supercoiled dsDNA.
-Transformation done to protocol for all gRNAs into DH5?. Full volume (250?l) plated onto Amp  plates and  incubated o/n.
+Transformation done to protocol for all gRNAs into DH5α. Full volume (250μl) plated onto Amp  plates and  incubated o/n.
 Week 10
 /16
@@ Line 3,024: / Line 1,138: @@
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+p51 (1)	62.5	1.90	1.65
-!  Sample
+     p51(2)	87.2	1.95	2.20
-! ng/ ?L
+p51(3)	112.7	1.81	1.34
-! 260/280
+p51(4)	67.1	1.92	2.24
-! 260/230
+sc1 (1)	126.5	1.83	1.48
+sc1(2)	101.9	1.88	1.84
-|-
+_t
-| p51 (1)
-| 62.5
-| 1.90
-| 1.65
-|-
-|     p51(2)
-| 87.2
-| 1.95
-| 2.20
-|-
-| p51(3)
-| 112.7
-| 1.81
-| 1.34
-|-
-| p51(4)
-| 67.1
-| 1.92
-| 2.24
-|-
-| sc1 (1)
-| 126.5
-| 1.83
-| 1.48
-|-
-| sc1(2)
-| 101.9
-| 1.88
-| 1.84
-|-
-|}
 Restriction digest done to protocol:
-{| class = "wikitable"
+_t Sample	Buffer Green Fast Digest	Enzyme Mix*	DNA
-|-
+(200 ng)	MQ H2O	Total
-!  Sample
+Volume
-! Buffer Green Fast Digest
+p51 (1)	1.0 μL 	1.0 μL	3.0 μL	5.0 μL	10.0 μL
-! Enzyme Mix*
+p51 (2)	1.0 μL 	1.0 μL	2.0 μL	6.0 μL	10.0 μL
-! DNA
+p51 (3)	1.0 μL	1.0 μL	2.0 μL	6.0 μL	10.0 μL
+p51 (4)	1.0 μL	1.0 μL	3.0 μL	5.0 μL	10.0 μL
-|-
+SC1 (1)	1.0 μL	1.0 μL	2.0 μL	6.0 μL	10.0 μL
-| (200 ng)
+SC1 (2)	1.0 μL	1.0 μL	2.0 μL	6.0 μL	10.0 μL
-| MQ H2O
+_t
-| Total
+*Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL BgI1 + 23.0 μL mqH2O
+Samples evaporated after digesting to a total volume of around 4 μL
-|-
-| Volume
-|-
-| p51 (1)
-| 1.0 ?L
-| 1.0 ?L
-| 3.0 ?L
-| 5.0 ?L
-| 10.0 ?L
-|-
-| p51 (2)
-| 1.0 ?L
-| 1.0 ?L
-| 2.0 ?L
-| 6.0 ?L
-| 10.0 ?L
-|-
-| p51 (3)
-| 1.0 ?L
-| 1.0 ?L
-| 2.0 ?L
-| 6.0 ?L
-| 10.0 ?L
-|-
-| p51 (4)
-| 1.0 ?L
-| 1.0 ?L
-| 3.0 ?L
-| 5.0 ?L
-| 10.0 ?L
-|-
-| SC1 (1)
-| 1.0 ?L
-| 1.0 ?L
-| 2.0 ?L
-| 6.0 ?L
-| 10.0 ?L
-|-
-| SC1 (2)
-| 1.0 ?L
-| 1.0 ?L
-| 2.0 ?L
-| 6.0 ?L
-| 10.0 ?L
-|-
-|}
-*Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L BgI1 + 23.0 ?L mqH2O
-Samples evaporated after digesting to a total volume of around 4 ?L
 Run on a 1% Gel. The following lists the sample loaded into each lane.
-{| class = "wikitable"
+_t Lane	Sample
-|-
+	2-Log DNA Ladder
-!  Lane
+	SDM p51 (1)
-! Sample
+	SDM p51 (3)
+	SDM SC1 (1)
-|-
+	SDM SC1 (2)
-| 1
+	Blank
-| 2-Log DNA Ladder
+	SDM P51 (4)
+_t
-|-
-| 2
-| SDM p51 (1)
-|-
-| 3
-| SDM p51 (3)
-|-
-| 4
-| SDM SC1 (1)
-|-
-| 5
-| SDM SC1 (2)
-|-
-| 6
-| Blank
-|-
-| 7
-| SDM P51 (4)
-|-
-|}
 [[File:UCB-induced cc9-gel-140717.JPG]]
@@ Line 3,182: / Line 1,178: @@
 PCR amplify p50
-{| class = "wikitable"
+_t Reactants	Volume
-|-
+Q5 Master Mix 	12.5 μL
-!  Reactants
+μM forward primer 	1.5 μL
-! Volume
+μM reverse primer 	1.5 μL
+DNA p50 (1:50)	1.0 μL
-|-
+Nuclease Free H2O	9.0 μL
-| Q5 Master Mix
+Final Reaction Volume	25.0 μL
-| 12.5 ?L
+_t
-|-
-| 10 ?M forward primer
-| 1.5 ?L
-|-
-| 10 ?M reverse primer
-| 1.5 ?L
-|-
-| DNA p50 (1:50)
-| 1.0 ?L
-|-
-| Nuclease Free H2O
-| 9.0 ?L
-|-
-| Final Reaction Volume
-| 25.0 ?L
-|-
-|}
 /21/2014
@@ Line 3,218: / Line 1,191: @@
-{| class = "wikitable"
+_t Sample	Buffer cutsmart	Enzyme Xbal	Enzyme Spe1 	DNA
-|-
+(200 ng)	MQ H2O	Total
-!  Sample
+Volume
-! Buffer cutsmart
+p51 (1)	2.0 μL 	1.0 μL	1.0 μL	3.2 μL	12.8 μL	20.0 μL
-! Enzyme Xbal
+p51 (2)	2.0 μL 	1.0 μL	1.0 μL	            2.3 μL	13.7 μL	20.0 μL
-! Enzyme Spe1
+p51 (3)	2.0 μL	1.0 μL	1.0 μL	1.8 μL	14.2 μL	20.0 μL
-! DNA
+p51 (4)	2.0 μL	1.0 μL	1.0 μL	3.0 μL	13.0μL	20.0 μL
+SC1 (1)	2.0 μL	1.0 μL	1.0 μL	1.6 μL	14.4 μL	20.0 μL
-|-
+SC1 (2)	2.0 μL	1.0 μL	1.0 μL	2.0 μL	14.0μL	20.0 μL
-| (200 ng)
+P51	2.0 μL	1.0 μL	1.0 μL	4.0 μL	12.0μL
-| MQ H2O
+P51	2.0 μL	1.0 μL	1.0 μL	4.0 μL	12.0μL
-| Total
+p51	2.0 μL	1.0 μL	1.0 μL	4.0 μL	12.0μL
+_t
-|-
-| Volume
-|-
-| p51 (1)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 3.2 ?L
-| 12.8 ?L
-| 20.0 ?L
-|-
-| p51 (2)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-|             2.3 ?L
-| 13.7 ?L
-| 20.0 ?L
-|-
-| p51 (3)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 1.8 ?L
-| 14.2 ?L
-| 20.0 ?L
-|-
-| p51 (4)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 3.0 ?L
-| 13.0?L
-| 20.0 ?L
-|-
-| SC1 (1)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 1.6 ?L
-| 14.4 ?L
-| 20.0 ?L
-|-
-| SC1 (2)
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 2.0 ?L
-| 14.0?L
-| 20.0 ?L
-|-
-| P51
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 4.0 ?L
-| 12.0?L
-|
-|-
-| P51
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 4.0 ?L
-| 12.0?L
-|
-|-
-| p51
-| 2.0 ?L
-| 1.0 ?L
-| 1.0 ?L
-| 4.0 ?L
-| 12.0?L
-|
-|-
-|}
 Enzymes are used as a 1:25 dilution
@@ Line 3,327: / Line 1,214: @@
 Sent p51 SDM 1, 3, 4 and p51 original as a control for sequencing. Confirmation that SDM was successful and p51 no longer has the restriction site spe1 in the gRNA location.
-Sequencing: Total volume 0f 15 ?L
+Sequencing: Total volume 0f 15 μL
-{| class = "wikitable"
+_t	 SDM Product	DNA μL	Primer p51seq5	MQ H2O
-|-
+	SDM1p51	8.0μL	2.5 μL	4.5μL
-!
+	SDM3p51	4.4μL	2.5μL	8.1μL
-!  SDM Product
+	SDM4p51	7.5μL	2.5μL	5.0μL
-! DNA ?L
+	p51 control	4.2μL	2.5μL	8.3μL
-! Primer p51seq5
+_t
-! MQ H2O
-|-
-| 1
-| SDM1p51
-| 8.0?L
-| 2.5 ?L
-| 4.5?L
-|-
-| 2
-| SDM3p51
-| 4.4?L
-| 2.5?L
-| 8.1?L
-|-
-| 3
-| SDM4p51
-| 7.5?L
-| 2.5?L
-| 5.0?L
-|-
-| 4
-| p51 control
-| 4.2?L
-| 2.5?L
-| 8.3?L
-|-
-|}
 Mini prepped 10 o/n cultures of SDM of p50. Done to protocol. Glycerol stocks make of all 10 cultures.  Mini prepped previous gibson colonies 1 and 2.  SDM of p51 for all samples were confirmed
   Nanodrops were taken to assess the concentration and purity of the samples:
-{| class = "wikitable"
+_t Sample	ng/ μL	260/280	260/230
-|-
+p50	123.7	1.84	2.15
-!  Sample
+     SDM1	179.6	1.88	2.19
-! ng/ ?L
+SDM2	117.4	1.90	2.21
-! 260/280
+SDM3	140.4	1.84	1.81
-! 260/230
+    SDM4	162.6	1.89	2.22
+SDM5	117.3	1.89	2.00
+SDM6	176.4	1.88	2.01
+SDM7	177.0	1.87	2.30
+SDM8	250.1	1.89	2.25
+SDM9	298.3	1.90	2.31
+SDM10	136.0	1.91	2.17
+Gibson1	110.8	1.89	2.14
+Gibson2	160.3	1.87	2.15
+ _t
-|-
+Restriction digest done to protocol:
-| p50
-| 123.7
-| 1.84
-| 2.15
-|-
+_t Sample	Buffer Cutsmart	Enzyme Mix*	DNA (200ng)	MQ H2O	Total Volume
-|      SDM1
+SDM p50 (1)	2.0 μL 	1.0 μL	1.2 μL	15.8 μL	20.0 μL
-| 179.6
+SDM p50 (2)	2.0 μL 	1.0 μL	2.0 μL	15.0 μL	20.0 μL
-| 1.88
+SDM p50 (3)	2.0 μL	1.0 μL	1.4 μL	15.6 μL	20.0 μL
-| 2.19
+SDM p50  (4)	2.0 μL	1.0 μL	1.2 μL	15.8μL	20.0 μL
+SDM p50  (5)	2.0 μL	1.0 μL	2.0 μL	15.0 μL	20.0 μL
+SDM p50 (6)	2.0 μL	1.0 μL	1.2 μL	15.8 μL	20.0 μL
+SDM p50 (7)	2.0 μL	1.0 μL	1.2 μL	15.8μL	20.0 μL
+SDM p50 (8)	2.0 μL	1.0 μL	1.0 μL	16.0μL	20.0 μL
+SDM p50 (9)	2.0 μL	1.0 μL	1.0 μL	16.0μL	20.0 μL
+SDM p50 (10)	2.0 μL	1.0 μL	1.5 μL	15.5μL	20.0 μL
+p50control	2.0 μL	1.0 μL	2.0 μL	15.0μL	20.0 μL
+_t
+*Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL Xba1 + 48.0 μL mqH2O
-|-
+Restriction Digest for gibson done to protocol:
-| SDM2
+_t Sample	Buffer D	Enzyme EcoRV	DNA
-| 117.4
-| 1.90
-| 2.21
-|-
-| SDM3
-| 140.4
-| 1.84
-| 1.81
-|-
-|     SDM4
-| 162.6
-| 1.89
-| 2.22
-|-
-| SDM5
-| 117.3
-| 1.89
-| 2.00
-|-
-| SDM6
-| 176.4
-| 1.88
-| 2.01
-|-
-| SDM7
-| 177.0
-| 1.87
-| 2.30
-|-
-| SDM8
-| 250.1
-| 1.89
-| 2.25
-|-
-| SDM9
-| 298.3
-| 1.90
-| 2.31
-|-
-| SDM10
-| 136.0
-| 1.91
-| 2.17
-|-
-| Gibson1
-| 110.8
-| 1.89
-| 2.14
-|-
-| Gibson2
-| 160.3
-| 1.87
-| 2.15
-|-
-|  _t
-|-
-|
-|-
-| Restriction digest done to protocol:
-|-
-|
-|-
-|}
-SDM p50 (1)	2.0 ?L 	1.0 ?L	1.2 ?L	15.8 ?L	20.0 ?L
-SDM p50 (2)	2.0 ?L 	1.0 ?L	2.0 ?L	15.0 ?L	20.0 ?L
-SDM p50 (3)	2.0 ?L	1.0 ?L	1.4 ?L	15.6 ?L	20.0 ?L
-SDM p50  (4)	2.0 ?L	1.0 ?L	1.2 ?L	15.8?L	20.0 ?L
-SDM p50  (5)	2.0 ?L	1.0 ?L	2.0 ?L	15.0 ?L	20.0 ?L
-SDM p50 (6)	2.0 ?L	1.0 ?L	1.2 ?L	15.8 ?L	20.0 ?L
-SDM p50 (7)	2.0 ?L	1.0 ?L	1.2 ?L	15.8?L	20.0 ?L
-SDM p50 (8)	2.0 ?L	1.0 ?L	1.0 ?L	16.0?L	20.0 ?L
-SDM p50 (9)	2.0 ?L	1.0 ?L	1.0 ?L	16.0?L	20.0 ?L
-SDM p50 (10)	2.0 ?L	1.0 ?L	1.5 ?L	15.5?L	20.0 ?L
-p50control	2.0 ?L	1.0 ?L	2.0 ?L	15.0?L	20.0 ?L
-{| class = "wikitable"
-|-
-!
-|-
-| *Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L Xba1 + 48.0 ?L mqH2O
-|-
-|
-|-
-| Restriction Digest for gibson done to protocol:
-|-
-|}
 (200 ng)	MQ H2O	Total
 Volume
-Gibson 1	2.0 ?L 	1.0 ?L	2.0 ?L	15.0 ?L	20.0 ?L
+Gibson 1	2.0 μL 	1.0 μL	2.0 μL	15.0 μL	20.0 μL
-Gibson 2	2.0 ?L 	1.0 ?L	1.2 ?L	15.8 ?L	20.0 ?L
+Gibson 2	2.0 μL 	1.0 μL	1.2 μL	15.8 μL	20.0 μL
-{| class = "wikitable"
+_t
-|-
+*EcoRv dilution (1:25)
-!
-!
-!
-|-
-| *EcoRv dilution (1:25)
-|-
+Run on a 1% Gel. The following lists the sample loaded into each lane.
-|
+_t Lane	Sample
-|-
-|
-|-
-| Run on a 1% Gel. The following lists the sample loaded into each lane.
-|-
-|}
 	2-Log DNA Ladder
 	SDM p50 (1)
@@ Line 3,537: / Line 1,288: @@
 	blank
-{| class = "wikitable"
+_t
-|-
+[[File:UCB-induced cc9-gel-140723.JPG]]
-!
-|-
-| [[File:UCB-induced cc9-gel-140723.JPG]]
-|-
+/25/2014
-|
-|-
+Restriction digest done to protocol:
-|
-|-
+_t Sample	Buffer Cutsmart	Enzyme Mix*	DNA
-| 7/25/2014
-|-
-|
-|-
-| Restriction digest done to protocol:
-|-
-|
-|-
-|}
 (250 ng)	MQ H2O	Total
 Volume
-SDM p50 (1)	2.0 ?L 	1.0 ?L	1.5 ?L	20.0 ?L	25.0 ?L
+SDM p50 (1)	2.0 μL 	1.0 μL	1.5 μL	20.0 μL	25.0 μL
-SDM p50 (2)	2.0 ?L 	1.0 ?L	2.0 ?L	19.5 ?L	25.0 ?L
+SDM p50 (2)	2.0 μL 	1.0 μL	2.0 μL	19.5 μL	25.0 μL
-SDM p50 (3)	2.0 ?L	1.0 ?L	1.8 ?L	19.7 ?L	25.0 ?L
+SDM p50 (3)	2.0 μL	1.0 μL	1.8 μL	19.7 μL	25.0 μL
-SDM p50  (4)	2.0 ?L	1.0 ?L	1.5 ?L	20.0?L	25.0 ?L
+SDM p50  (4)	2.0 μL	1.0 μL	1.5 μL	20.0μL	25.0 μL
-SDM p50  (5)	2.0 ?L	1.0 ?L	2.0 ?L	19.5 ?L	25.0 ?L
+SDM p50  (5)	2.0 μL	1.0 μL	2.0 μL	19.5 μL	25.0 μL
-SDM p50 (6)	2.0 ?L	1.0 ?L	1.5 ?L	20.0 ?L	25.0 ?L
+SDM p50 (6)	2.0 μL	1.0 μL	1.5 μL	20.0 μL	25.0 μL
-SDM p50 (10)	2.0 ?L	1.0 ?L	2.0 ?L	19.5?L	25.0 ?L
+SDM p50 (10)	2.0 μL	1.0 μL	2.0 μL	19.5μL	25.0 μL
-p50control	2.0 ?L	1.0 ?L	2.0 ?L	19.5?L	25.0 ?L
+p50control	2.0 μL	1.0 μL	2.0 μL	19.5μL	25.0 μL
-{| class = "wikitable"
+_t
-|-
+*Enzyme mix: 5.5μL Xba1 + 5.5μL Spe1 for a total volume of 11μL
-!
-!
-!
-|-
-| *Enzyme mix: 5.5?L Xba1 + 5.5?L Spe1 for a total volume of 11?L
-|-
-|
-|-
-| Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.
-|-
-|
-|-
+Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.
-| Restriction Ligation
-|-
+Restriction Ligation
-|
-|-
+_t Sample	Buffer Cutsmart	EcoR1	Xba1	Spe1	Pst1	DNA
-|}
 (200 ng)	MQ H2O	Total
 Volume
-SDM p50 (1)	5.0 ?L 	--	1.0 ?L	--	1.0 ?L	1.0 ?L	42.0 ?L	50.0 ?L
+SDM p50 (1)	5.0 μL 	--	1.0 μL	--	1.0 μL	1.0 μL	42.0 μL	50.0 μL
-SDM p50 (4)	5.0 ?L 	--	1.0 ?L	--	1.0 ?L	      1.0 ?L	42.0 ?L	50.0 ?L
+SDM p50 (4)	5.0 μL 	--	1.0 μL	--	1.0 μL	      1.0 μL	42.0 μL	50.0 μL
-SDM p50 (6)	5.0 ?L	--	1.0 ?L	--	1.0 ?L	1.0 ?L	42.0 ?L	50.0 ?L
+SDM p50 (6)	5.0 μL	--	1.0 μL	--	1.0 μL	1.0 μL	42.0 μL	50.0 μL
-Litimus28i	5.0 ?L	--	--	1.0 ?L	1.0 ?L	1.5 ?L	41.5?L	50.0 ?L
+Litimus28i	5.0 μL	--	--	1.0 μL	1.0 μL	1.5 μL	41.5μL	50.0 μL
-Litimus+Ic3	5.0 ?L	1.0 ?L	--	--	1.0 ?L	0.8 ?L	42.2 ?L	50.0 ?L
+Litimus+Ic3	5.0 μL	1.0 μL	--	--	1.0 μL	0.8 μL	42.2 μL	50.0 μL
-PsBGA1 (RFP)	5.0 ?L	1.0 ?L	--	1.0 ?L	--	3.8 ?L	39.2 ?L	50.0 ?L
+PsBGA1 (RFP)	5.0 μL	1.0 μL	--	1.0 μL	--	3.8 μL	39.2 μL	50.0 μL
-{| class = "wikitable"
+_t
-|-
-!
-|-
+Sequenced SDMp50 was not the correct product, the above project did not continue.
-|
-|-
+/18/2014
-| Sequenced SDMp50 was not the correct product, the above project did not continue.
+O/N cultures of MG, Bw+Kan, Bw-kan
-|-
+/19/2014
-|
+O/N cultures were all made competent according to protocol
+O/D recordings:
-|-
+_t Time	MG	Bw+kan	Bw(-)kan
-| 8/18/2014
-|-
-| O/N cultures of MG, Bw+Kan, Bw-kan
-|-
-|
-|-
-| 8/19/2014
-|-
-| O/N cultures were all made competent according to protocol
-|-
-| O/D recordings:
-|-
-|
-|-
-|}
 :00	.042	.026	.071
 :34	.086	.035	.088
@@ Line 3,652: / Line 1,343: @@
 :10	--	.308	--
 :15	--	.320	--
-{| class = "wikitable"
+_t
-|-
-!
-|-
-|
-|-
-| The cells were all pulled at the time they were at an O/D  nearest .325. All O/D’s were diluted to .30
-|-
-| Once the cells were made competent they were transformed using a gRNA  target, gRNA scramble, and  a sb1c3  strain.
-|-
-|
-|-
+The cells were all pulled at the time they were at an O/D  nearest .325. All O/D’s were diluted to .30
-{{Template:UCB-Footer}}
+Once the cells were made competent they were transformed using a gRNA  target, gRNA scramble, and  a sb1c3  strain.