Team:Paris Bettencourt/Notebook/Odor Library
From 2014.igem.org
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</ol> | </ol> | ||
</div> | </div> | ||
- | <strong>Protocol: Heat Shock Transformation of <em>E. coli</em></strong | + | <strong>Protocol: Heat Shock Transformation of <em>E. coli</em></strong> |
- | 1) Place 20 ul of cells in a pre-chilled Eppendorf tube.< | + | 1) Place 20 ul of cells in a pre-chilled Eppendorf tube.</br>2) Add 1 ul of DNA to the chilled cells</br> |
- | + | 3) Mix gently by flicking the tube.</br>4) Chill on ice for 10 minutes.<br/> | |
- | + | 5) Heat shock at 42 °C for 30 seconds.<br/> | |
- | 5) Heat shock at 42 °C for 30 seconds.<br | + | 6) Return to ice for 2 minutes.<br/> |
- | 6) Return to ice for 2 minutes.<br | + | 7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.Another rich medium can substitute for the recovery.<br/>The recovery time varies with the antibiotic selection.</br> |
- | 7) Add 200 ul LB medium and recover the cells by shaking at 37 °C. | + | Tetracycline: 60-120 minutes</br>Chloramphenicol: 60-120 minutes</br> |
- | + | 8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate. </br>Use glass beads to spread the cells. | |
- | 8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate. <br | + | |
- | + | <h1>6/06 Transformations</h1> | |
- | <h1 | + | |
<div>Yesterday's plates do not have colonies. </div> | <div>Yesterday's plates do not have colonies. </div> | ||
<div><br clear="none"/></div> | <div><br clear="none"/></div> |
Revision as of 17:06, 15 October 2014
Notebook
June
June 26th
Goal
Design plasmid for limonene synthase
Procedure
Used MG1665 strain, the RBS with the highest initial rate and the sequence of r-limonene synthase from genBank. Added polyhistidine tail to the construct. Submitted the construct to Jake.
Results
Designed plasmid for limonene synthase using the software geneious. Found a biobricks part containing this sequence. For the purpose of saving budget, we would first transform this standard part into cell and later modify it.
June 27th
Goal
Transform the bioBricks limonene synthase(BBa_I742111) part into e.coli. We found that the bioBricks kit for 2014 contain the limonene synthase sequence in plate 4 position 3I.
Procedure
- Start thawing the competent cells(Used Christina's competent cells) on ice.
- Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
- Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
- Close tubes and incubate the cells on ice for 30 minutes.
- Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
- Incubate the cells on ice for 5 minutes.
- Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
- Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
- For the control, label two petri dishes with LB agar (CM). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
- You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Results
Transformed the plasmid containing limonene synthase sequence from biobricks into E.Coli. Colonies seen on both plates. Glycerol Stock made: PB. 018 and PB. 019
June 28th
Goal
Made chemical competent cell following standard protocol with MG1665 strains.
Procedure
- The night before, inoculate a 5 ml culture and grow overnight with selection.
- The day of the experiment dilute cells ~ 1:200 into selective media. For this example add 250 ul to 50 ml of selective media. Note: The protocol is easily scaled to increase the number of cells.
- Grow the cells to an OD600 of 0.6 – 0.7. Use a large flask, 500ml, for good aeration. Use a baffled flask for fastest growth. This takes about 3 hours depending on the cells. Medium-heavy cloudiness by eye is fine.
- Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
- Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
- Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
- Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
- Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
Note: Frozen cells are only good once.Do not refreeze cells once thawed.
Results
The transformed cell were grown for 20 hours and the transformation was successful. The plates were put into the 4 degree fridge for making stocks on Monday.
July
July 1st
Goal
To design the primers and gBlock to modify the BioBricks part BBa_I742111.
Procedure
- decide the major steps of modification
- to PCR the biobricks construct using primers that have Agel restriction site sequences.
- to digest the PCR product
- to PCR the gBlock with Promoter, RBS and restriction sites (Xbal and Agel)
- to digest the PCR product
- to ligate the two sequences together
- 2. Design the primers for PCR and the gBlock including promoter, RBS and restriction sites using the software Geneious.
Results
- fwd primer for BioBricks part PCR: 0PB.015
- rev primer for BioBricks: 0PB.016
- gBlock with promoter, RBS and restriction sites: 0PB. 019
- fwd primer for gBlock PCR: 0PB.30
- rev primer for gBlock PCR: 0PB.31
- (We ended up ordering the gBlock as synthetic gene sequence, because it is too long for oligos and too short for gBlocks. Oh well.)
July 7th
Goal
to extract plasmid of biobricks I742111 from the transformed cells.
Procedure
- Growth the cell for two days
- Centrifuge the tube for 15 minutes with 4000 rps
- Follow the standard protocol of Thermo Scientific miniprep kit
- Label the centrifuge tube and store it in the -20 freezer. (Syl miniprep)
Results
- We grew two tubes of glycerol stocks. However living cells were only seen in one of them, which was used for miniprep.
July 8th
Goal
Amplify limonene synthase gene sequence for E.coli
- Fw primer: oPB.015
- Rv primer: oPB.016
Procedure
Reagent | Volume |
1x | |
Nuclease-free water | 71 ul |
5x Phusion HF Buffer | 20 ul |
10 mM dNTPs | 2 ul |
Forward Primer (10 uM) | 1 ul |
Reverse Primer (10 uM) | 1 ul |
Template Plasmid | 1 ul |
Phusion DNA Polymerase | 1 ul |
DMSO | 3 ul |
Total Volume | 100 ul |
Temperature | Time | Function | |
start | 98 C | 30 sec | melt |
cycle 1 | 98 C | 10 sec | melt |
cycle 2 | 51 C | 30 sec | anneal |
cycle 3 | 72 C | 30 sec | extend |
finish | 72 C | 5 min | extend |
blind | 10 C | forever | blind |
Results
PCR product at the expected length.
July 9th
Goal
- To run the Gel of the PCR product to confirm that the segment is at the right length.
- to digest the segment using AgeI and XbaI. Purify the digested product.
Procedure
- Take the PCR tubes out of the machine.
- Mixing the dye with PCR products, from each tube.
- Run the gel for an hour and half using 100bp plus DNA ruler
- Observe under UV light
- Digest in the 37 degree incubator for 10 minutes using AgeI and XbaI.
- Purify following the protocol of digestion purification kit. Used warm NF water for diluting DNA at the last step.
Reagent Volume Nuclease-free water 27.5 ul 10x FD Buffer 5 ul DNA 12.5 ul AgeI 2.5 ul Xbal 2.5 ul Total Volume 50 ul
Results
The PCR product is at expected length. Purified product has a very low concentration(2.5ng/ul), meaning the PCR product was a mixture of multiple segments and didn't give ideal result. Therefore proceed to PCR again.
July 16th
Goal
Amplify limonene synthase gene sequence for E.coli
- Fw primer: oPB.015
- Rv primer: oPB.016
Procedure
Reagent | Volume |
1x | |
Nuclease-free water | 71 ul |
5x Phusion HF Buffer | 20 ul |
10 mM dNTPs | 2 ul |
Forward Primer (10 uM) | 1 ul |
Reverse Primer (10 uM) | 1 ul |
Template Plasmid | 1 ul |
Phusion DNA Polymerase | 1 ul |
DMSO | 3 ul |
Total Volume | 100 ul |
Temperature | Time | Function | |
start | 98 C | 30 sec | melt |
cycle 1 | 98 C | 10 sec | melt |
cycle 2 | 51 C | 30 sec | anneal |
cycle 3 | 72 C | 2.5 min | extend |
finish | 72 C | 5 min | extend |
blind | 10 C | forever | blind |
Results
It worked!
![](https://static.igem.org/mediawiki/2014/1/1d/Sylviagel1.png)
Goal
: transform the PB19 synthase part into e.coli.
Procedure
- Start thawing the competent cells(Used Christina's competent cells) on ice.
- Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
- Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
- Close tubes and incubate the cells on ice for 30 minutes.
- Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
- Incubate the cells on ice for 5 minutes.
- Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
- Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
- For the control, label two petri dishes with LB agar (CM). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
- You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Results
Colonies seen on both plates.
July 17th
Goal
to extract plasmid of PB19 from the transformed cells.
Procedure
- Growth the cell overnight
- Centrifuge the tube for 15 minutes with 4000 rps
- Follow the standard protocol of Thermo Scientific miniprep kit
- Label the centrifuge tube and store it in the -20 freezer. (Syl mini prep)
Results
- Tube 1: 40 ng/ul
- Tube 2: 30ng/ul
Goal
to digest the segment using AgeI and XbaI. Purify the digested product.
Procedure
Digest in the 37 degree incubator for 10 minutes using AgeI and XbaI.
Reagent | Volume |
Nuclease-free water | 27.5 ul |
10x FD Buffer | 5 ul |
DNA | 12.5 ul |
AgeI | 2.5 ul |
Xbal | 2.5 ul |
Total Volume | 50 ul |
Purify following the protocol of digestion purification kit. Used warm NF water for diluting DNA at the last step
Results
Concentration after purification: 12.2 ng/ul
July 23rd
Goal
: to achieve the great great goal of magnifying the RBS and promoter before digestion, purification and ligation.
Procedure
we did a nano drop of the mini prep plasmid, and it is 460ng/ul. The curve looks good. We diluted 1:500
![](https://static.igem.org/mediawiki/2014/5/56/Sylviaimg2.jpeg)
Results
a fainted band was seen. However, the concentration after purification is below 5 ng/ul, too low to be used to ligation. Therefore, we decided to design the sequence as two oligos, with Xbal and Agel sticky ends.
August
August 4th
Goal
to dilute received oilgos, to Phosphorylate them, to Anneal them and to ligation is with Limonene vector
Procedure
- Dilute the oilgo to 100 uM, the stock
- Take 10 ul out of the 100 uM stock and dilute to make 10 uM working stock
- Mix:
- 2 uL 10 µM sense oligo
- 2 uL 10 µM anti-sense oligo
- 2 uL 10 x PNK (polynucleotide kinase) buffer A
- 2 uL 10mM ATP
- 1 uL T4 polynucleotide kinase (PNK)
- 10 uL distilled water
- to give 20 uL total volume
- Incubate at 37C for 30 mins
- Place in boiling water bath for 2 min, then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature. (Labelled Annealing in the box)
- ligation
- Mix :
- 8 ul of vector (12 ng/ul)
- 1 ul of insert
- 0.5 ul of ligase enzyme
- 2 ul of ligase buffer
- 8.5 ul of distilled water
- total 20 ul
- put under 22 degree for 30 minutes
- put under 16 degree for 30 minutes
- put in 4 degree overnight
Results
colonies were seen after transforming and plating.
August 10th
Goal
to sequence the ligated plasmid for limonene construct
Procedure
- Dilute the mini prep product 1: 2 (Give us concentration 70ng/ul and 80ng/ul)
- design primers that are about 200 bp away upstream and downstream from the inserted sequence
- Send them to GATC
Results
It appears that the old plasmid recirculate itself.
Further Steps
regrow 6 more colonies, minipreped and did analytical PCR with NotI and AgeI
Colony 1a appears to have the complete plasmid.
August 20th
Goal
to PCR ilvBN gene from MG1655
Procedure
- Picked 2 colonies from the plate MG1655 115+GFP
- Inoculate them each in 50 ul of NF H2O
- Boiled for 3 mins at 98 degree
- Use the solution as DNA template
- Follow standard PCR protocol from this step on
Reagent | Volume |
1x | |
Nuclease-free water | 63 ul |
5x Phusion HF Buffer | 20 ul |
10 mM dNTPs | 2 ul |
Forward Primer (10 uM) | 5 ul |
Reverse Primer (10 uM) | 5 ul |
Template Plasmid | 1 ul |
Phusion DNA Polymerase | 1 ul |
DMSO | 3 ul |
Total Volume | 100 ul |
Temperature | Time | Function | |
start | 98 C | 30 sec | melt |
cycle 1 | 98 C | 10 sec | melt |
cycle 2 | 51 C | 30 sec | anneal |
cycle 3 | 72 C | 2 min | extend |
finish | 72 C | 5 min | extend |
blind | 10 C | forever | blind |
Results
Band seen on both sample (from 2 colonies)
August 21st
Goal
to Digest the PCR product (ilvBN) from yesterday and to digest pET-DUET1 as vector
Procedure
Vector (pET-DUET1)
Reagent | Volume |
Nuclease-free water | 38 ul |
10x FD Buffer | 5 ul |
DNA | 2 ul |
EcoRI | 2 ul |
NcoI | 2 ul |
FastAP | 1 ul |
Total Volume | 50 ul |
ilvBN
Reagent | Volume |
Nuclease-free water | 39 ul |
10x FD Buffer | 5 ul |
DNA | 2 ul |
EcoRI | 2 ul |
NcoI | 2 ul |
Total Volume | 50 ul |
Incubate at 37 degree for 2 hours. Purification using the PCR purification kit.
Results
24 ng/ul for C2 and 12 ng/ul for C1 (Will use C2 PCR purified product for digestion.)
August 25th
Goal
to verify the insert of RBS and promoter is in the limonene biobrick part.
Procedure
digest
Reagent | Volume |
Nuclease-free water | 19 ul |
10x FD Buffer | 3 ul |
DNA | 1 ul |
AgeI | 1 ul |
Xbal | 1 ul |
Total Volume | 25 ul |
(Miniprep concentration: 500 for N.1, 400 for N.2 and N.3) Expected length: 3750 and 3740 or 3840 and 3740
Results
(with 100bp plus DNA ladder)
![](https://static.igem.org/mediawiki/2014/0/01/Sylviaimg5.png)
(the control sample didn’t get digested. Therefore, we can’t say for sure if the ligation worked. However, we decided to go ahead with sequencing and see the result.)
August 26th
Goal
To ligate ilvBN gene with PETDUET 1
Procedure
Reagent | Volume |
Nuclease-free water | 3 ul |
Ligase Buffer | 2 ul |
ligase | 1 ul |
Insert(2132 bp, 10ng/ul) | 4 ul |
Vector(5381bp, 18ng.ul) | 10 ul |
Total Volume | 20 ul |
25 degree for 1 hour and then heat shock transformation.
Results
colonies seen. Picked 4 colonies for analytical digestion. Bands seen at expected length.
August 27th
Goal
to ligate the aldB gblock with pSB1C3
Procedure
Digested and Purified PCR product, concentration: 40 ng/ul Digested and purified vector pSB1C3: 20 ng/ul
Reagent | Volume |
Nuclease-free water | 10 ul |
Ligase Buffer | 2 ul |
ligase | 1 ul |
Insert(2132 bp, 10ng/ul) | 4 ul |
Vector(5381bp, 18ng.ul) | 4.5 ul |
Total Volume | 20 ul |
20 degree for 5 hours, followed by heat shock transformation into NEB strain.
Results
Blurring band seen on gel after colony PCR, will process to mini prep and sequencing
5/06 Odourless coli
5/06 Banana and Wintergreen E. coli Transformation
- pPB.003 BBa_R0040 2013 3 5E pSB1C3
- pPB.004 BBa_J45199 2013 4 6I pSB1C3
- pPB.005 BBa_J45119 2012 2 5B pSB1AT3
- Resuspend DNA from registry plates using 10 uL of ddH2O. Make sure to resuspend GOOD.
- Follow protocol below. Using 1 uL of DNA.
5) Heat shock at 42 °C for 30 seconds.
6) Return to ice for 2 minutes.
7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.Another rich medium can substitute for the recovery.
The recovery time varies with the antibiotic selection. Tetracycline: 60-120 minutesChloramphenicol: 60-120 minutes 8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate. Use glass beads to spread the cells.
6/06 Transformations
- Plate the 200 uL from yesterday's tube containing recovered cells.
-
- pPB.003 BBa_R0040 LBA+Cm
- pPB.004 BBa_J45199 LBA+Cm
- pPB.005 BBa_J45119 LBA+Tet
- pPB.003 BBa_R0040 LBA+Cm
- Transform:
-
- pPB.003 BBa_R0040: Constitutive TetR promoter
-
- Resuspended DNA from 2013:3:5E
- pSB1C3 Chloramphenicol resistance
- pPB.004 BBa_J45199: Banana generator without promoter
-
- Resuspended DNA from 2013:4:6I
- pSB1C3 Chloramphenicol resistance
- Requires promoter and 5mM isoamyl alcohol
- pPB.005 BBa_J45119: Wintergreen generator without promoter
-
- Resuspended DNA from 2012:2:5B
- pSB1AT3 Ampicillin and Tetracycline resistance
- Requires promoter and 2 mM Salicylic acid
- USED, NOT AVAILABLE BBa_J45200: Constitutive Banana generator
-
- 2012:2:5F
- pSB1AT3 Ampicillin and Tetracycline resistance
- Requires 5mM isoamyl alcohol
- USED, NOT AVAILABLE BBa_J45120: Constitutive Wintergreen generator
-
- 2012:2:5D
- pSB1AT3 Ampicillin and Tetracycline resistance
- Requires 2 mM Salicylic acid
- Plate fresh transformations:
-
- pPB.003 BBa_R0040 LBA+Cm
- BBa_J45119 LBA+Tet
- BBa_J45199 LBA+Cm
- Streak NEB Turbo in LBA for singles (tomorrow set an overnight for competent cell production).
7/06 Single colonies from Transformations |
8/06 Single colonies from Transformations |
9/06 Single colonies from Transformations |
No growth. Picked single colonies and started overnights again.
10/06 Minipreps |
26/06 Digestion set up |
Digestion:
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
17
|
Buffer |
3
|
FD Enzyme I |
1
|
FD Enzyme II |
1
|
DNA |
3
|
Total Volume |
25
|
- 2.1 kb
- 18 bp
- ~ 2 kb
- 1.765 kb
- 3.4 kb
- 1.26 kb
27/06 SAGE set up |
- Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
- Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
- Max amount is 400 mg
- Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
- Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
- Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
- Place column in a 2 mL collection tube
- Load sample into column. Centrifuge 1 min @ 13 000 rpm.
- Discard flowthrough and repeat adding 0.5 mL of QG buffer.
- Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
- Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
- Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
- Store at 4ºC overnight or -20ºC for longer periods.
![](19.-%20Wiki%20notebook.resources/Pasted_Image_6_30_14_11_54_AM-48.jpg)
27/06 Ligation set up |
7/07 We start again... |
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
17
|
Buffer |
3
|
FD Enzyme I |
1
|
FD Enzyme II |
1
|
DNA |
3
|
Total Volume |
25
|
- 2.1 kb
- 18 bp
- ~ 2 kb
- 1.765 kb
- 3.4 kb
- 1.26 kb
14/07 Digestion-product gel extraction and ligation |
- 2.1 kb
- 18 bp
- ~ 2 kb
- 1.765 kb
- 3.4 kb
- 1.26 kb
17/07/2014 Restriction Digestion set up |
Digestion:
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
75
|
Buffer |
10
|
FD Enzyme I |
2.5
|
FD Enzyme II |
2.5
|
DNA |
10
|
Total Volume |
100
|
- 2.1 kb
- 18 bp
- ~ 2 kb
- 1.765 kb
- 3.4 kb
- 1.26 kb
- ~2.1 kb
- ~18 kb
21/07/2014 Ligation set up |
-
Fermentas T4 DNA Ligation ReagentVector (pPB.003)25 ng Insert75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL
Fermentas T4 DNA Ligation ReagentVector2 uL Insert2 uL NF ddH2O4.5 uL T4 DNA Ligase Buffer1 uL T4 DNA Ligase0.5 uL Total10 uL - Incubate @22ºC for 10 minutes.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
pPB.016 Banana Generator under Anderson Promoter |
- pPB.016: Banana generator AndersonPromoter (BBa_J23108)
-
- Requires 5mM isoamyl alcohol
7/07 Transformations |
- pPB.014 BBa_J23108 2014 4 4C pSB1C3
- Resuspend DNA from registry plates using 10 uL of ddH2O. Make sure to resuspend GOOD.
- Follow protocol below. Using 1 uL of DNA.
1) Place 20 ul of cells in a pre-chilled Eppendorf tube.
2) Add 1 ul of DNA to the chilled cells
5) Heat shock at 42 °C for 30 seconds.
6) Return to ice for 2 minutes.
7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.Another rich medium can substitute for the recovery.
The recovery time varies with the antibiotic selection.
Chloramphenicol: 60-120 minutes
8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate.
Use glass beads to spread the cells.
- Transform:
- Constitutive Promoter
-
- BBa_J23108: Constitutive Promoter from Anderson's Library
-
- Resuspended DNA from 2014:4:4C
- pSB1C3 Chloramphenicol resistance
- BBa_J45199: Banana generator without promoter
-
- Resuspended DNA from 2013:4:6I
- pSB1C3 Chloramphenicol resistance
- Requires promoter and 5mM isoamyl alcohol
1) Place 20 ul of cells in a pre-chilled Eppendorf tube.
2) Add 2 ul of DNA to the chilled cells
5) Heat shock at 42 °C for 30 seconds.
6) Return to ice for 2 minutes.
7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.Another rich medium can substitute for the recovery.
The recovery time varies with the antibiotic selection.
Chloramphenicol: 60-120 minutes
8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate.
Use glass beads to spread the cells.
- Plate fresh transformations:
-
- BBa_R0040 LBA+Cm
- BBa_J45119 LBA+Tet
- BBa_J45199 LBA+Cm
- Streak NEB Turbo in LBA for singles (tomorrow set an overnight for competent cell production).
- Send email asking for salicylic acid and isoamyl alcohol.
8/07 Single colonies from Transformations |
8/07 Minipreps |
8/07 Digestion from miniprep set up |
Digestion:
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
17
|
Buffer |
3
|
FD Enzyme I |
1
|
FD Enzyme II |
1
|
DNA |
3
|
Total Volume |
25
|
- ~2 kb
- ~18 bp
- ~ 2 kb
- 1.765 kb
8/07 SAGE set up |
- Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
- Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
- Max amount is 400 mg
- Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
- Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
- Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
- Place column in a 2 mL collection tube
- Load sample into column. Centrifuge 1 min @ 13 000 rpm.
- Discard flowthrough and repeat adding 0.5 mL of QG buffer.
- Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
- Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
- Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
- Store at 4ºC overnight or -20ºC for longer periods.
Ligation set up |
-
Fermentas T4 DNA Ligation ReagentVector (pPB.003)25 ng Insert (pPB.014)75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL - Incubate vector @22ºC for 60-120 minutes and insert for 0.5-1 hour.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
- Recover for 60 minutes on LB.
- Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
- Recovered the cells for 45 minutes: No colonies (27/06)
- Re-plated remaining recovered cells (overnight @ room temperature): No colonies (27/06)
- Repeated transformation (29/06): No colonies.
pPB.016 Enhanced Wintergreen Generator |
- pPB.015: Enhanced Wintergreen generator Salis Promoter (BBa_23108)
-
- Requires 2 mM Salicylic acid
8/07 Digestion from miniprep set up
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
17
|
Buffer |
3
|
FD Enzyme I |
1
|
FD Enzyme II |
1
|
DNA |
3
|
Total Volume |
25
|
- ~2 kb
- ~18 bp
- 3.4 kb
- 1.26 kb
8/07 SAGE set up |
- Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
- Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
- Max amount is 400 mg
- Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
- Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
- Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
- Place column in a 2 mL collection tube
- Load sample into column. Centrifuge 1 min @ 13 000 rpm.
- Discard flowthrough and repeat adding 0.5 mL of QG buffer.
- Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
- Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
- Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
- Store at 4ºC overnight or -20ºC for longer periods.
Ligation set up |
-
Fermentas T4 DNA Ligation ReagentVector (pPB.014)25 ng Insert (pPB.005)75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL - Incubate @22ºC for 10 minutes.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
- Recover for 60 minutes on LB.
- Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
- No colonies.
- Run gel of old samples
-
- pPB.003 TetR Promoter
- pPB.004 RBS-ATF1
- pPB.005 RBS-BMST1
- pPB.014 BBa_J23108
- Start 5 mL overnights of single colonies from Estefania’s plates.
-
- pPB.015: Anderson’s Promoter + BMST1
- pPB.016: Anderson’s Promoter + ATF1
- pPB.022 (BBa_J45004) : BMST1 from Registry 2012 kit
- pPB.003 (Digested with SpeI & PstI) ~2.1 kb
- pPB.004 (Digested with XbaI & PstI) ~1.765 kb
- pPB.005 (Digested with XbaI & PstI) ~1.26 kb
- pPB.014 (Digested with SpeI & PstI) ~2 kb
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
17
|
Buffer |
3
|
FD NheI |
1
|
FD PstI |
1
|
DNA |
3
|
Total Volume |
25
|
-
Fermentas T4 DNA Ligation ReagentVector (pPB.003 or pPB.014)25 ng Insert (pPB.004 or pPB.005)75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL - Incubate @22ºC for 10 minutes.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
- Recover for 60 minutes on LB.
- Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
- No colonies.
- ~2 kb
- ~18 bp
- ~ 2 kb
- 1.765 kb
FD Restriction Digestion
|
|
Reagent | Volume (uL) |
NF ddH2O |
13.5
|
Buffer |
4
|
FD Enzyme I |
1.5
|
FD Enzyme II |
1.5
|
DNA |
3
|
Fast AP | 1.5 uL |
-
Fermentas T4 DNA Ligation ReagentVector (pPB.003)25 ng Insert75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL - Incubate @22ºC for 10 minutes.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
- Recover for 60 minutes on LB.
- Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
![](19.-%20Wiki%20notebook.resources/Screen%20Shot%202014-09-23%20at%209.36.24%20AM.png)
![](19.-%20Wiki%20notebook.resources/Screen%20Shot%202014-09-23%20at%209.45.02%20AM.png)
![](19.-%20Wiki%20notebook.resources/Screen%20Shot%202014-09-23%20at%209.58.39%20AM.png)
Reagent | Volume | |
1x | 4x | |
Nuclease-free water | 71 ul | 284 ul |
5x Phusion HF Buffer | 20 ul | 80 ul |
10 mM dNTPs | 2 ul | 8 ul |
Forward Primer (10 uM) | 1 ul | 4 ul |
Reverse Primer (10 uM) | 1 ul | 4 ul |
Template DNA (Plasmid, 1:10 dilution) | 1 ul | 4 ul |
DMSO | 3 ul | 12 ul |
Phusion DNA Polymerase | 1 ul | 4 ul |
Total Volume | 100 ul | |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 52 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 40 sec | Extend | |
Finish | 72 °C | 5 min | Extend | |
Store | 10 °C | Forever | Store |
Keep insert+vector volume below 4 uL.
Fermentas T4 DNA Ligation ReagentVector (pPB.003)25 ng Insert75 ng T4 DNA Ligase Buffer 10X1 uL T4 DNA Ligase0.5 uL NF-ddH2Oas needed Total10 uL - Incubate @22ºC for 10 minutes.
- Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
- Recover for 60 minutes on LB.
- Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 52 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
![](19.-%20Wiki%20notebook.resources/gBlocks_5_and_6_pSB1AT3-44.jpg)
![](19.-%20Wiki%20notebook.resources/sm133_fam.jpg)
I will repeat the PCR for the gPB.003 (oPB.013 and oPB.014), gPB.005, gPB.006 (JW.340 and JW.341) using the following conditions:
Phusion MasterMix | |
Reagent | Volume (uL) |
DNA | 1 |
Fwd (10 uM) | 2.5 |
Rev (10 uM) | 2.5 |
DMSO | 1.5 |
MM | 25 |
H2O | 17.5 |
50 |
I will do each sample by duplicate varying only the [DNA template] using 1 uL directly from stock tubes and a 1:10 dilution as well. For the pSB1C3 (oPB.081 and oPB.082) I will use 1 uL of pPB.003 (RBS) or pPB.004 (Banana generator) at 1:10 and 1:100 dilutions.
![](19.-%20Wiki%20notebook.resources/Digestion_of_gBlocks-15.jpg)
![](19.-%20Wiki%20notebook.resources/PCR_58-14.jpg)
![](19.-%20Wiki%20notebook.resources/gel_day_1-2.jpg)
![](19.-%20Wiki%20notebook.resources/Gel_day_2-4.jpg)
Phusion MasterMix | Final concentration | |
Reagent | Volume (uL) | |
DNA |
1
|
< 250
|
Fwd (10 uM) |
1
|
0.5 uM
|
Rev (10 uM) |
1
|
0.5 uM
|
DMSO |
0.6
|
3%
|
MM |
10
|
1X
|
H2O |
6.4
|
|
20
|
Fermentas T4 DNA Ligation | |
Reagent
|
|
Vector (pPB.003)
|
25 ng |
Insert
|
75 ng |
T4 DNA Ligase Buffer 10X
|
1 uL |
T4 DNA Ligase
|
0.5 uL |
NF-ddH2O
|
as needed |
Total
|
10 uL |
Digestion of vectors | |
Plamid DNA |
20
|
10x FD Buffer |
10
|
FD Enzyme I |
2.5
|
FD Enzyme II |
2.5
|
FastAP |
2.5
|
NF H2O |
62.5
|
Total |
100
|
Digestion of inserts | |
Plamid DNA |
16
|
10x FD Buffer |
2
|
FD Enzyme I |
1
|
FD Enzyme II |
1
|
NF H2O |
0
|
Total |
20
|
Phusion MasterMix | Final concentration | |
Reagent | Volume (uL) | |
DNA |
1
|
< 250
|
Fwd (10 uM) |
1
|
0.5 uM
|
Rev (10 uM) |
1
|
0.5 uM
|
DMSO |
0.6
|
3%
|
MM |
10
|
1X
|
H2O |
6.4
|
|
20
|
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 55 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 50 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 59 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
![](19.-%20Wiki%20notebook.resources/gBlocks_PCR_chida-29.jpg)
- 300 bp
- 968 bp
- 1258 bp
- 2076 bp
Banana BioBrick
|
||||
[DNA] (ng/uL) | Volume (uL) | Amount (ng) | ||
vector pPB.014 |
34
|
1
|
34
|
|
insert (pPB.004) |
15
|
5
|
75
|
|
Buffer |
2
|
|||
T4 Ligase |
1
|
|||
9
|
||||
H2O |
11
|
|||
Jasmine gBlock
|
||||
[DNA] (ng/uL) | Volume (uL) | Amount (ng) | ||
vector |
4
|
12
|
48
|
|
insert (pPB.003) |
5
|
19
|
92.4
|
1307
|
Buffer |
6
|
|||
T4 Ligase |
3
|
|||
40
|
Phusion MasterMix | Final concentration | |
Reagent | Volume (uL) | |
DNA |
1
|
< 250
|
Fwd (10 uM) |
1
|
0.5 uM
|
Rev (10 uM) |
1
|
0.5 uM
|
DMSO |
0.6
|
3%
|
MM |
10
|
1X
|
H2O |
6.4
|
|
20
|
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 59 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
Phusion MasterMix | Final concentration | |
Reagent | Volume (uL) | |
DNA |
1
|
< 250
|
Fwd (10 uM) |
1
|
0.5 uM
|
Rev (10 uM) |
1
|
0.5 uM
|
DMSO |
0.6
|
3%
|
MM |
10
|
1X
|
H2O |
6.4
|
|
20
|
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 50.7 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 60 sec | Extend | |
Finish | 72 °C | 10 min | Extend | |
Store | 10 °C | Forever | Store |
Reagent | Qty |
5x M9 Salts | 200 mL |
1M MgSO4 | 2 mL |
20% Glucose Stock Solution |
20 mL |
1M CaCl2 | 0.1 mL |
Complete AA mix | 2 g |