Team:Cooper Union/Notebook/Telomere

6/3/14

(Nolana)

• Researched genes that affect telomere length (lengthening or shortening).
• Possible genes to be used for knockout: HSP104 (long), MAK31 (long), VPS75 (short), SMI1 (short).
• Relevant lit: PNAS_Askree

For tomorrow: see if yeast plasmids in lab can be used for programmable kill switch project. Design primers for gene deletion protocol.

6/4/14

(Nolana)

• Designed primers for yeast genes (TLC1, RAD52, HSP104, VPS75, MAK31, SMI1).
• Checked plasmids to be ordered for knockout cassettes (pFA6a-kanMX6, pFA6a-TRP1, pAG25).
• Transformed DH5α with 6 different BioBrick plasmids (pSB1A3, pSB1C3, pSB1K3, pSB1T3, pSB2K3), two BioBrick RBS (B0030, B0034), and a BioBrick double terminator (B0015); left all 8 to grow overnight.

For tomorrow: check for cell growth on plates and further grow any colonies that form.

6/5/14

(Nolana)

• Plates were checked; colonies were growing on all of them.
• Picked 4 genes to order primer oligos for (RAD52, EST4, MAK31, VPS75).
• Streaked plates with old yeast strains.

For Monday (I won't be here tomorrow): check yeast plates for colonies

6/9/14

(Nolana)

• Checked yeast plates -- all plates grew colonies.
• Since EST4 knocked out in conjunction with RAD52 kills the yeast, we decided to use EST1 instead of EST4 in the gene deletion for the kill switch project.
• Poured plates -- 3 bottles of amp (100ug/mL). We used 250 μL of 100 mg/mL stock concentration of Ampicillin.
• Poured two 0.8% agarose gels, both about 60 mL in each.
• Helped set up Restriction Digest Reaction with De novo group (used General Restriction Enzyme Digest protocol): 3 μL buffer, 3 μL BSA, DNA, H₂O, and Xho1/Xba1 enzyme.

For tomorrow: design check primers for knockout cassettes (programmable kill switch project)

6/10/14

(Nolana)

Designed check primers to be used to verify gene deletion -- used oligo JM42 (3' tag sequence of sense orientation deletion primer with homology to MX4 cassette) for reverse primer, and 500 bases upstream of each gene (RAD52, EST1, MAK31, VPS75) for forward primer.

For tomorrow: Design amplifying primers for kill switch project.

6/11/14

(Nolana)

• Worked with Devora to find the primer sequences for amplifying the VPS75, RAD52, MAK31, EST1; added BioBrick prefixes and suffixes.
• Checked all of the different primers and sequences that we ordered to check for forbidden restriction sites: there is one restriction site in KanMX, one in LEU2, and 3 forbidden restriction sites in TRP1; these will need to undergo PCR mutagenesis in order to remove the restriction sites.
• made YPD plates: (meant to make three 250mL bottles of media, but ended up making one 500mL bottle)

bacto-yeast extract (1%)	5 g
bacto-peptone (2%)	10 g
glucose (2%)	25 mL
bacto-agar (2%)	10 g
distilled H2O	500 mL

For tomorrow: make more YPD plates!

6/12/14

(Nolana)

Made three 250mL bottles of Synthetic Complete (SC) and Dropout Media for selectable YPD plates (1 without leucine, 1 without tryptophan, 1 without leucine+tryptophan):

bacto-yeast nitrogen base	1.675 g
glucose (2%)	12.5 mL
bacto-agar (2%)	5 g
dropout mix (0.2%)	0.5 g
distilled H2O	232 mL

Nitrogen base was without amino acids.
Dropout mix was without histidine, leucine, tryptophan, uracil.

Constituents to add to media as needed:

• 2.5 mL leucine
• 0.5 mL histidine
• 0.5 mL tryptophan
• 2.5 mL uracil

6/13/14

(Nolana)

• Checked if plasmids in the freezer can be used in kill switch project (they can). We have pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP.
• Streaked out plates of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP, put them in 37 degrees incubator to grow.
• Made glucose 40% with 200g dextrose and distilled water to make 500mL of solution (two 250mL bottles).
• Made clonat plates: regular YPD plates+clonat
• Tried to shift primers for gene deletion protocol because of forbidden restriction sites.

6/16/14

(Nolana)

• Researched linearizing the genome for E. coli; found that the method described in EMBO_Cui may not be ideal for the kill switch project.
• Helped De novo group start their double restriction digest.
• Discussed expanding upon yeast system to make it so that the yeast strains programmed with the kill switch cannot mate with wild-type yeast strains.
• Inoculated the cultures of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP and left them growing overnight in LB broth with Amp antibiotic.

For tomorrow: Isolate pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP plasmids, read into yeast background papers.

6/17/14

(Nolana)

• Checked the cells grown overnight; confirmed the correct plasmids had been grown (pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP)
• Prepared 1mL miniculture of equal part cells and a 60% glycerol stock for each sample to be stored in the -80C freezer.
• miniprepped the rest of the overnight cultures (2 samples of each plasmid), following the Miniprep Protocol with some exceptions. The 5mL of overnight culture was centrifuged at 3900rpm for 10 minutes for the first step of preparing the cell lysate. For the second step of eluting the DNA, 50μl of preheated TE buffer was added to the column instead of the stated 75μl.
• After the plasmids were isolated, the concentration of each sample was measured using the nanodrop, then stored at -20°C.
• Helped Dionne nanodrop her samples.

Nanodrop results:

pFA6a-kanMX6-PGAL1-GFP

601.4 ng/μl

725.8 ng/μl

pFA6a-GFP(S65T)-TRP1

364.9 ng/μl

438.2 ng/μl

For tomorrow: go over yeast background papers to find yeast mating gene for deletion.

6/18/14

(Nolana)

• Plasmids and primers arrived today (yay!)
• Streaked out plasmids onto Amp plates (pAG25, pFA6a-LEU2MX6, pFA6a-TRP1)
• Reconstituted primers and made 100x umol stocks of primers with TE buffer
• Worked with Devora to label and dilute all of the 100 uM conc. of the primers; dilution was 1:10 so the diluted primers should be a 10 umolar concentration (did 10 uL resuspended primer with 90 uL ddH2O).



List of Primers:

EST1 deletion (forward and reverse)

RAD52 deletion (forward and reverse)

MAK31 deletion (forward and reverse)

VPS75 deletion (forward and reverse)

EST1 BioBrick (forward and reverse)

RAD52 BioBrick (forward and reverse)

De Novo group: TdT (forward and reverse)

De Novo group: PolyA (forward and reverse)



• Colony PCR amplified LEU2, TRPI, and pAG25 (we got this by stabbing the colony within the ring in the glass bottles that were ordered and swirling that into 200 uL of ddH2O water) and for each we did 2 sets of PCR; 1 with ESTI deletion primers and the other with RAD52 deletion primers. We did 20 uL of each of the plasmid along with 2.5 uL each of the forward and reverse primers. As a control we ran 20 uL of ddH2O water with 2.5 each of the primers (so a control for both EstI and Rad52). We added an extension time of 2 minutes. We left that to PCR overnight because the machine is heat-regulated so it's fine if it stays in there.

For tomorrow: check PCR

6/19/14

(Nolana)

• Ran gels of PCR samples with Jaeho on two 0.8% gels, using a 1kb BioLabs ladder. The wells can be read as [ladder, EST1 (E), EST1+TRP1 (ET), EST1+pAG25 (EC), EST1+LEU2 (EL), ladder] for the first gel, and [ladder, RAD52 (R), RAD52 +TRP1 (RT), RAD52 +pAG25 (RC), RAD52 +LEU2 (RL), ladder] for the second gel (see attached images).
• Checked gels after 30 minutes. Primers were confirmed to be fine, but for both EST1 and RAD52, only the TRP1 amplifiers worked.
• Left gels to run for another 30 minutes, but there was negligible changes.

• Troubleshooting:

  template problem: instead of using colony PCR with bacteria, miniprep plasmids first

  PCR conditions: maybe use extension time of 3 minutes instead of 2; maybe lower self annealing temperature from 55 to 52.

  possible contaminants in sample (unlikely because the TRP1 amplifier worked fine)

  check sequences of clonat (pAG25) and LEU2

  Sizes expected on gels:

  EST1: 127 bp

  ET: 1074 bp

  EC: 1358 bp

  EL: 2415 bp

  RAD52: 127 bp

  RT: 1074 bp

  RC: 1358 bp

  RL: 2415 bp

• Prepared for yeast transformation: made one 250mL bottle of YPD liquid media, colony PCR'ed EST+TRP1, made yeast cultures of W303a and W303alpha.
  YPD liquid media:

  yeast extract (1%)	2.5g
  peptone (2%)	5g
  glucose (40%)	12.5 mL
  distilled H2O	237.5 mL

• Colony PCR:

  took bacteria from pFA6a-TRP1 plasmid bottle and swirled in 200 uL H2O

  added 20 uL TRP1, 2.5 uL of EST1 forward and reverse primers into 4 PCR tubes

  used PCR program 333

• yeast culture:

  took W303a and W303alpha colonies and added to ~20 mL YPD (2%)

  put samples in shaker (37 degrees, 250 rpm)

  For tomorrow: check PCR, do yeast transformation

6/20/14

(Nolana)

• Finished another 250mL bottle of liquid YPD media by adding 12.5mL glucose.
• Worked with Lily and Jaeho to take a look at the yeast cultures we inoculated yesterday (W303a and W303alpha). The W303alpha seemed to be contaminated with bacteria, so we started counting the number of cells per mL of yeast in the W303a culture. We used a hemacytometer to count, and determined that if we used that culture to do a yeast transformation, the yield would be very low (ideal number of cells/mL is 5 million, we had around 2.5 million).
• We abandoned the W303a culture, deciding to do a new culture of the strain, this time from newer plates (the ones we grew yesterday were taken from old plates).
• Looked at newer colonies of W303a under a microscope, the yeast on the new plates look healthy.
• Purified PCR product from yesterday (EST1+TRP1) and separated into two 200 uL samples.
• After purification, the concentration of each sample was measured using the nanodrop, then stored at -20°C.
  

  Nanodrop results:

  EST1+TRP1(1): 81.4 ng/uL

  EST1+TRP1(2): 77.9 ng/uL

For tomorrow: yeast transformation (for real this time)

6/21/14

(Nolana)

• Looked at newly inoculated yeast cells (W303a, W303alpha) under microscope. The cells were were round and budding, meaning they were healthy and not contaminated.
• Worked with Jaeho and Lily to count cells. The original yeast cultures had too many cells to count, so we used a 110uL 1:10 dilution of W303alpha and a 100uL 1:100 dilution of W303a.

  Average number of cells:

  W303a: 1,355,000 cells/mL

  W303alpha: 2,020,500 cells/mL

• Calculated ratios of YPD and culture to make a cell density of 5x10⁶ cells/mL.
• Inoculated cultures with YPD and put in shaker at 3pm.
  

  W303a: 10.25mL culture + 14.7mL YPD = 25 mL total

  W303alpha: 9.9mL culture + 4mL YPD = 13.9mL total
  Note: total should have been 25mL, but we thought we did not have enough culture. But actually, we did the math wrong and had to redo the inoculation, finally putting the cultures into the shaker at 5pm, to be checked at 7:30.

  W303a: 6.5x10⁷ cells/mL ----> 2.5mL culture + 25mL YPD = 27.5 mL total

  W303alpha: 4.5x10⁷ cells/mL ----> 2.5mL culture + 22.5mL YPD = 25mL total

• Yeast cells were ready at 8:45pm, with cell density ~2x10^7
• Followed Small-scale LiAc Yeast Transformation Procedure up to step 21, where Jae and I plated the yeast (which had EST1+TRP1 added to them) on TRP- plates, along with two negative controls (W303a and W303alpha without EST1+TRP1). The plates were left in the 30 degree incubator to sit for 2-3 days.

6/23/14

(Nolana)

• Poured LB Amp plates with Wilfrido because the lab was running out.
• Made two 0.8% gels for Shoshana's experiment.
• Made a 6% gel for the De Novo group's CleanAmp TdT experiment which will be run tomorrow.

For tomorrow: check yeast selective plates and continue transformation

6/24/14

(Nolana)

• Checked yeast selective plates: there were ~4 colonies per plate, but that was including the negative controls, which should not have any colonies (possible mutation in yeast strain?)
• Worked with Lily to streak out plates for each colony (4 W303a +TRP1 plates, 4 W303a neg. con. plates, 3 W303alpha+TRP1 plates, 4 W303 neg. con. plates), and put all 15 plates in 30 degree incubator. These plates were split into quadrants, where each day ~12:30pm, we will check the colony growth and restreak in the next quadrant. Each quadrant, to be checked daily, goes through ~20 divisions. Hopefully we will see the growth of the colonies fade and come back, following the growth curve of an EST1 knockout.
• Poured YPD plates (three 250mL bottles)
• Colony PCR'ed EST1+LEU2 and RAD52+LEU2: what we changed from the last PCR was the annealing temperature to 54 degrees instead of 55, and the extension time from 2 minutes to 3 minutes, using PCR program 333.
• Inoculated plasmids for tomorrow's miniprep (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6, pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP).

For tomorrow: miniprep plasmids, check yeast TRP-selective plates

6/25/14

(Nolana)

• Worked with Jaeho to miniprep plasmids (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6)
• Ran gel of PCR samples (EST1+LEU2 and RAD52+LEU2) on a 0.8% gel. There were no results. Need to check if primer sequences are correct.
• Nanodropped plasmids from miniprep:

  pAG25 (clonat): 563.7 ng/uL

  pFA6a-TRP1: 455.7 ng/uL

  pFA6a-LEU2MX6: 799.7 ng/uL

• Checked why PCR gel didn't work: we looked at the primer sequences but there was no problem.
• We made a 1:1000 dilution of each plasmid (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6), then ran another PCR, this time with the purified plasmids from the miniprep.
  

  5 uL plasmid

  15 uL H2O

  2.5 uL each of the EST1 deletion primers (forward and reverse)

  PCR conditions were the same as last time, program 333.

For tomorrow: check PCR and run a gel; check on yeast selective plates.

6/26/14

(Jae Ho)
PCR Check Done, Gel run.
2-3 kbp results for EST1+LEU2, no results on other. Possibly contaminated template of Clonet.
Digest and run gel of clonet to check if it is contaminated etc
Yeast cultures are struck and photos taken.


For later: Digest/Gel Clonet, make more YPD plates (20 more)


(Nolana)

• made YPD plates with Lily
• looked up enzymes and buffer for clonat double digest: we will use the enzyme sites SacI and HindIII, and the NEB Cutsmart buffer

For Monday: run digest

6/30/14

(Nolana)

• Looked at yeast plates -- not very conclusive
• Ran clonat double digest:

  Cutsmart buffer	2 uL
  10x BSA	2 uL
  pAG25	1 uL
  ddH2O	13 uL
  SacI	1 uL
  BamHI-HF	1 uL
  Total Volume: 20 uL

• Designed BioBrick primer for EST2 (for yeast strains Professor Medvedik brought back from Boston)
• Streaked 5 YPD plates with yeast strains and put them in 30 degree incubator: W303alpha, 1300, 1294, 1296, 1297
• Helped Wilfrido make two 0.8% gels, and two 1% gels
• Ran a 0.8% gel. Wells: 1. Versa Ladder ; 2. digested clonat ; 3. undigested clonat ; 4-7. Shoshana's samples
• Gel results: the SacI cut at 43bp, and the BamHI-HF at 1280, which gave two pieces of 1237bp and 2467bp, confirmed by the gel. The undigested clonat result also looked good. This confirmed that the plasmid we have is what we want, the pAG25 (clonat). Now, the question is why didn't it work with the deletion primers?
• Made yeast cultures of W303a, 1880, 1882 for yeast transformation tomorrow

For tomorrow: run PCR of MAK31 and VPS75; yeast transformation

7/1/14

(Nolana)

• Yeast transformed inoculated overnight cultures of 1880, 1882, and W303a
• PCR'd 4 samples for yeast deletion cassettes: MAK31+TRP1 (MT), MAK31+LEU2 (ML), VPS75+TRP1 (VL), VPS75+LEU2 (VL)
• Took photos of yeast plates to check for growth (1294, 1296, 1297, 1300, and W303alpha); restreaked all plates

(Jae Ho)

Yeast Transformation>>Follows Protocol
PCR plasmid purification skipped due to time restraints.
AMT, AVT, 82VL, 80ML Transformations prepared, and plated. w303A, 1882, 1880 Negative control plates also prepared.

For tomorrow: run gel of PCR samples

7/2/14

(Nolana, Jae Ho)

• restreaked yeast plates from last week (the ones that were likely to be TRP);
  6 Plates restreaked at day 4 after day 1 (faint): W303alpha+TRP1-1, W303alpha negative control, W303alpha+TRP1-3
• Photos of yeast plates/restreaked: 1300, 1294, 1296,1297, w303alpha
  Due to deletion of EST2, 1294,1296,1297 are fading:dying
• New transformation done by prof. Medvedik: wrong plates on original transformation
• Ran gel with yesterday's PCR samples (MT, ML, VT, VL) with one of the De Novo group's pet28+TdT samples. Used VersaLadder.
  Wells: 1. blank ; 2. ladder ; 3. VT ; 4. VL ; 5. MT ; 6. ML ; 7. pET28+TdT
  

  Sizes expected: 1074 for VT and MT, 2415 VL and ML

• Results: we had to stain the gel with ethidium bromide because everything was too faint at first.
  VL and ML had approximately 2kb; VT and MT had approximately 1kb --> good results!
  However, there were other bands that showed along with the PCR amplification -- maybe raise temperature to 56 instead of 54.

For tomorrow: run PCR with same samples, but with different temperature

7/3/14

(Jae Ho)
PCR machine not available, unable to do PCR
Restreaked cultures and took photoes
Restreaked successful transformation colonies: Mak31+TRP1/VPS75+TRP1

For later: PCR EST1+LEU2, EST1+TRP1
Out of TRP plates

7/7/14

PCR: EST1+TRP1, EST1+LEU2
Culture preps: W303A, W303alpha, 1300, 1296
New YPD plates made
Plates struck
Gel Run with 1% gel and 1k ladder: Two showed nothing while the third ET showed multiple bands.
Could be contamination in sample/annealing temperature problem (54->56, maybe try 58)

Tomorrow: Transformation

7/8/14

(Nolana)

• Ran 1% gel of PCR samples ET1, ET2, EL1, ET2. Wells: 2. BioLabs 1kb ladder ; 3. EL1 ; 3. EL2 ; ET1 ; ET2
• Gel results were faint, so we stained the gel in ethidium bromide for 15 minutes.
  

  Expected sizes -- ET: 1074 ; EL: 2415.

  Results: the ET sizes matched but the EL did not -- maybe the plasmid template is contaminated;

  miniprep LEU2 plasmid again?

• Checked and restreaked yeast plates to check for growth
• Yeast transformation of W303alpha, W303a, 1300, 1296
• 1296 discarded for transformation
• Knockout of EST1+TRP1/LEU2 for other strains in progress

For tomorrow: Miniprep TRP, LEU
Check MAK31 and VPS75 knock plates by colony PCR
Make TRP and LEU plates

7/9/14

(Lily)

• Checked and restruck yeast plates
• Left culture overnight for miniprep tomorrow (will do!)

(Nolana)

• Made TRP- and LEU- selective yeast plates
• helped Dionne run her PCR samples on a 1.5% gel (wells: 1. VersaLadder ; 2-7:(1)-(6) ; 8. (8) )
• ran PCR overnight (colony PCR for 2 samples each of MAK31::TRP1, MAK31::LEU2, VPS75::TRP1, VPS75::LEU2 and 2 samples each of ET, EL)

For tomorrow: re-miniprep plasmids for pFA6a-TRP1, pFA6a-LEU3MX6;
Run gel for colony PCR samples
Purify PCR samples of knockout cassettes
Check and restreak yeast plates

7/10/14

(Nolana)

• Miniprepped pFA6a-TRP1 and pFA6a-LEU2MX6 plasmid templates

• Nanodropped miniprep samples:

  TRP1: 298.4 ng/uL

  LEU2: 163.8 ng/uL

• Yeast plates were restruck and photos were taken for growth monitoring
• PCR'ed two samples each of EST1+LEU2, EST1+TRP1 for PCR purification and then yeast transformation tomorrow.
  PCR conditions: program 333, temperature 54, 180 seconds extension time, 30 cycles
• Ran two 1% gels of yesterday's PCR product:
  

  gel 1 wells: 1-3: blank ; 4. MT1 ; 5: ML1 ; 6. VT1 ; 7. VL1 ; 8. Biolabs 100bp ladder

  gel 2 wells: 1-3: blank ; 4. MT2 ; 5: ML2 ; 6. VT2 ; 7. VL2 ; 8. Biolabs 100bp ladder

• Inoculated yeast strains in YPD for overnight culture for transformation

For tomorrow: purify PCR products and the yeast transform strains 1300, W303A, W303alpha

7/11/14

(Jae Ho)
Transformation Not done due to lack of culture: Reculture on Sunday
PCR purification results: near 0 concentration: Redo PCR on Sunday
Gels ran again

gel 1 wells: 1blank ; 2. ladder 3. MT1 ; 4: ML1 ; 5. VT1 ; 6. VL1

gel 2 wells: 1blank ; 2. Ladder 3. MT2 ; 4: ML2 ; 5. VT2 ; 6. VL2

Plates restruck, photos taken

For Monday: Tranformation, PCR purification

7/14/14

PCR machine failure
Yeast plates restruck, photos taken
Overnight cultures of 1300. W303a. W303A remade
Gel run

gel 1 wells: 1blank ; 2. 100 base ladder 3. MT2 ; 4: ML1 ; 5. VT1 ; 6. VL1; 7 versa ladder

gel 2 wells: 1blank ; 2. 100 base ladder 3. MT2 ; 4: ML2 ; 5. VT2 ; 6. VL2 ; 7.versa ladder

Gel 2: the samples ran off so results were unobtainable.

Redoing PCR of EL and ET on the other machine.

Tomorrow: Check PCR (Gel/ purification), Transformation of 1300, W303a, W303A if PCR is successful.

7/15/14

• Checked gel pictures from last gel run --

• Expected sizes of gene w/out knockout:

  MAK31: 381 bp

  VPS75: 406 bp

  Expected sizes with knockout:

  MAK31: 285 bp

  VPS75: 275 bp

• Ran PCR of MT, ML, VT, VL (one regular, one universal sample) on the new OpenPCR machine, program 334:

  Initial Step: Temp 95, Time 300

  Anneal: Temp 56, Time 30

  Extend: Temp 72, Time 30

  Final Step: Temp 72, Time 420

  Final Hold: Temp 4

  Cycles: 30

• Ran two 1% gels (10uL PCR product, 2 uL 10x loading dye)
  

  Gel 1 wells: 1. blank ; 2. 100bp ladder ; 3. MT1 ; 4. ML1 ; 5. VT1 ; 6. VL1

  Gel 2 wells: 1. blank ; 2. 100bp ladder ; 3. MT2 ; 4. ML2 ; 5. VT2 ; 6. VL2

• Gel results: MAK31 and VPS75 did not get deleted from the yeast colonies (no bright bands on gel 2)
• Ran PCR again with same settings, but with new colonies

For tomorrow: run gel

07/16/14

• Made four 1% gels and ran them with the following:

  Colony 3: A-D

  Colony 4: E-G

• Ran gels of all PCR samples for genes without knockouts (samples A-H)

  Gel run

  gel 1.1 wells: 1blank ; 2. 250 base ladder ; 3. (A1) MT1 ; 4: (B1) ML1 ; 5. (C1) VT1 ; 6. (D1) VL1

  gel 1.2 wells: 1blank ; 2. 250 base ladder ; 3. (E1) MT2 ; 4: (F1) ML2 ; 5. (G1) VT2 ; 6. (H1) VL2

• Ran gels of all PCR samples for genes with universal primers (samples A-H)
  

  Gel run

  gel 2.1 wells: 1blank ; 2. 250 base ladder 3. (A2) MT1 ; 4: (B2) ML1 ; 5. (C2) VT1 ; 6. (D2) VL1

  gel 2.2 wells: 1blank ; 2. 250 base ladder 3. (E2) MT2 ; 4: (F2) ML2 ; 5. (G2) VT2 ; 6. (H2) VL2

• Results:

  gels 1.1 and 1.2 all had bands -- this indicates that the genes were not deleted

  gel 2.2 had no bands -- this goes along with the results from gel 1.1 and 1.2

  gel 2.1 had one very bright VT band -- streak out this colony to double check later?

For tomorrow: screen more colonies! If gels still show no deletions, then maybe do yeast transformation on Friday.

7/17/14

• Colony PCR

  Colony 5: A-D

  Colony 6: E-H

• Ran gels of all PCR samples for genes without knockouts (A-H 3)

Gel run

gel 1 wells: 1blank ; 2. 250 base ladder ; 3. (A3) MT1 ; 4: (B3) ML1 ; 5. (C3) VT1 ; 6. (D3) VL1

gel 2 wells: 1blank ; 2. 250 base ladder ; 3. (E3) MT2 ; 4: (F3) ML2 ; 5. (G3) VT2 ; 6. (H3) VL2

• Ran gels of all PCR samples for genes with universal primers (samples A-H 4)

  Gel run

  gel 3 wells: 1blank ; 2. 250 base ladder 3. (A4) MT1 ; 4: (B4) ML1 ; 5. (C4) VT1 ; 6. (D4) VL1

  gel 4 wells: 1blank ; 2. 250 base ladder 3. (E4) MT2 ; 4: (F4) ML2 ; 5. (G4) VT2 ; 6. (H4) VL2

• Results:

  bands for VPS75:: TRP1 for colonies 5 and 6 showed. possible knockout

• Restreaked VPS75:: TRP1 of colonies 3, 5, and 6

For tomorrow: screen more colonies! MAK31 colonies
Check restreaked colonies

7/18/14:

• Colony PCR'ed MAK31::TRP1 deletion strain. 10 samples: 5 samples from Plate A (A7-A11), 5 samples from Plate B (B1-B5).
  PCR settings: program 334, anneal temp: 56, extension time: 30 sec.
• Made two 1% agarose gels

• Ran gels

  gel 1 wells: 1. blank ; 2. Versa ladder ; 3-6. A7-10 ; 7-8. Shoshana and Devora's samples

  gel 2 wells: 1 blank ; 2. Versa ladder ; 3-7. B1-5 ; 8. A11

• Results: gel 1 had one faint band at A7, we stained the gel in 400 mL H2O and 500 uL ethidium bromide for 15 minutes.
  After staining, the band was still faint. All the Plate B samples on gel 2 had successful knockouts, but we realized the samples were taken from one streaked out colony.
• Streaked out 2 YPD plates for the MAK31:TRP1 deletion: 1 plate B colony, A7
• Overnight PCR of Plate B colony to double-check deletion: one sample with universal primer, one sample with gene check primer

For Monday: check the MAK31 colonies and the VPS colonies that were struck yesterday.
Prepare for new transformations for those strains with EST1.

7/21/14

• Made four 1% agarose gels
• ran PCR of VT colonies with possible knockouts that were streaked out last week. 6 samples: 2 of each colony (one with universal primer, one with gene check primer): colonies 3, 5, 6 using the OpenPCR program 334.
• Ran gel of last week's MT samples: 2 samples: one with universal primer, one with gene check primer. Wells: 1. blank ; 2. Versa ladder ; 3. universal ; 4. gene check
• Results: there were bands for both lanes on the gel, which corresponded to their respective sequence sizes (universal: 275, gene check: 406) -- maybe yeast strain is diploid?
• Ran gel of VT colonies. Wells: 1. Versa ladder ; 2. 3R ; 3. 5R ; 4. 6R ; 5. 3U ; 6. 5U ; 7. 6U
• Results: bands were faint, stained gel in 200mL H2O+250uL ethidium bromide. After staining, confirmed that 2 colonies had knockouts (3 and 6)
• Streaked out three plates of previous W303alpha transformation
• Streaked out original 1808 colony (pre-transformation)

For tomorrow: make more YPD plates, check MAK31 plates again, prepare for yeast transformation

7/22/14:

• Made YPD plates
• restreaked 1880, VPS75:TRP1 colonies 3, 6 for testing UV phenotype
• Ran overnight PCR samples of EST1+LEU2 for tomorrow (purification and yeast transformation
  

  Program: PCR for ~1-2kb 54c

  Initial Step: Temp 95, Time 300

  Denaturing: Temp 95, Time 30

  Anneal: Temp 54, Time 30

  Extend: Temp 72, Time 120

  Final Step: Temp 72, Time 420

  Final Hold: Temp 4

  Cycles: 30

• Prepared overnight cultures of MAK31:TRP1 and VPS75:TRP1 (3, 6)

For tomorrow: yeast transformation, PCR purification, miniprep LEU2

For some later date: make biobrick parts for VPS75 deletion

07/23/14

• Yeast transformation with EST1 of MAK31:TRP1 and VPS75:TRP1 (3, 6)
• Put dilution in shaker at 11:07 am
• PCR purified yesterday's samples (3 samples of EST1::LEU2)
• Checked MAK31 and VPS75 BioBrick primers for Prof. Medvedik to order
• Counted cells of MAK31 and VPS75

• Step 14 of transformation

  MT : EL1

  VT3 : EL2

  VT6 : EL3

• Streaked out 1880, VPS75:TRP1 (3, 6) on plate.
  Exposed to UV light on low setting for two seconds to confirm VPS75 phenotype (sensitivity to UV light)
• Inoculate a culture of LEU2 plasmid for miniprep tomorrow

7/24/14

• Miniprepped TRP and LEU

• Nanodrop results:

  TRP: 9.9 ng/uL

  LEU: 122.2 ng/uL

• prepared definitions and big ideas for meeting

7/28/14:

• Checked transformation plates: nothing grew except for the VPS75 Colony 3 plates, which had grown fungus because of a contamination in the YPD media
• Colony PCR'ed 3 samples of the first EST1 transformation with the universal primer to check for knockouts (OpenPCR program 334)
• Replicated 4 plates of 1880 strain+VPS75 knockouts. UV- shocked 3 plates on high: 10 sec, 30 sec, 1 min.
• Ran 1% gel of samples.

  Wells: 1. blank ; 2. Versa ladder 3. plate 6 ; 4. plate 7 ; 5 plate 8.

  Results: there were no bands on the gel, meaning that there were no knockouts.

  Expected sizes of EST1 knockout:

  with universal primer: 205 bp

  with gene check primer: 500 bp

• Inoculated cultures for tomorrow's transformation

For tomorrow: PCR EST1 deletion cassettes, purify PCR product, yeast transformation, check UV plates

7/29/14

• PCR'ed EST1+LEU2 for purification and transformation
• Put diluted yeast culture in shaker at 11:35am.
• Purified and nanodropped PCR product

  EL1: 9.2 ng/uL

  EL2: 4.8 ng/uL

  EL3: 2.7 ng/uL

  EL4: 13.6 ng/uL

• Transformed yeast strains VT3 + EL1, VT6 + EL2, MT7 + EL3, MTB + EL4

For tomorrow: make more LEU- plates

7/30/14

• Made more LEU- plates (1 bottle of 400 mL)
• Prepared for tomorrow's presentation
• Researched assaying protocol (x) (x) (x)
• Restreaked W303A and W303alpha for assaying on Friday (1:00 pm)

For tomorrow: finish presentation, make more YPD plates, prepare for assay

7/31/14

• Checked EST1 transformations: no growth on VT6 plate 1, MT7 plate 2, VT3 plate 1. There were non-yeast growths on MT7 plate 1 and VT3 plate 2
• Restreaked all 5 yeast colonies of VT3 plate 2
• Chose 2 colonies each for colony PCR: MTB plate 1, MTB plate 2, MT7 plate 1, VT6 plate 2
• Instead of using PCR tubes with beads, we used a standard PCR reaction:

  Master Mix	1x	9x
  10x ThermoPol Buffer	2.5 uL	22.5 uL
  Primers (Forward & Reverse)	5 uL	45 uL
  10mM dNTP	0.25 uL	2.25 uL
  Template	5 uL
  Taq DNA Polymerase	0.5 uL	4.5 uL
  ddH2O	11.75 uL	105.75
  Total	25 uL	180

• Poured YPD plates (two 250mL bottles)
• Made four 1% agarose gels
• Ran gels of PCR samples.

  Gel 1 Wells: 1. blank ; 2. 250bp ladder ; 3. MTB1(1) ; 4. MTB1(2) ; 5. MTB2(1) ; 6. MTB2(2).

  Gel 2 Wells: 1. Blank ; 2. 250bp ladder ; 3. MT71(1) ; 4. MT71(2) ; 5. VT62(1) ; 6. VT62(2)

  Gel Results

  we used too much ladder (wrong concentration).

  There were very faint bands -- maybe redo PCR with raised temperature.

  There were bright bands outside the ladder.

• Put 6mL inoculations of 1294, 1296, 1297, W303A, W303alpha into roller

For tomorrow: count yeast cells

8/1/14

• Checked yeast cells in roller
• counted cells with hemacytometer

  W303alpha: 1.31x10^8 cells/mL

  W303A: 1.4x10^8 cells/mL -- 5uL for dilution

  1296: 1.015x10^8 cells/mL -- 5uL for dilution

  1294: 2.805x10^6 cells/mL -- 214uL for dilution

  1297: 3.695x10^6 cells/mL -- Some of these yeast cells were dark and dying: this strain is in the middle of senescencing

• Diluted W303A, 1296, 1294 in triplicate in 6mL new YPD media to cell density of 10^5

For tomorrow: count cells and re-dilute

8/2/2014

Cell count:

1296

1: 6.4x10^7

2: 1.63x10^8

3: 1.28x10^8

Ave: 1.18x10^8+0.15-0.18

W303A

1: 3.08x10^7

2: 3.72x10^7

3: 2.99x10^7

Ave: 3.26x10^7+0.15-0.09

1294

1: 1.72x10^8

2: 1.63x10^8

3:1.83x10^8

Ave: 1.73x10^8+0.03-0.03

Recultured with 3-1-1: closest to average

1296: 3.906 ul

W303A: 16.26 ul

1294: 29.07 ul

8/3/2014

Cell count:

1296

1: 1.39x10^8

2: 1.39x10^8

3: 1.07x10^8

Ave: 1.28x10^8+0.04-0.07

W303A

1: 1.05x10^8

2: 9.2x10^7

3: 1.25x10^8

Ave: 1.07x10^8+0.06-0.05

1294

1: 1.41x10^8

2: 1.52x10^8

3:1.72x10^8

Ave: 1.55x10^8+0.06-0.05

Recultured with 1-2-1: closest to average

1296: 3.6 ul

W303A: 3.2 ul

1294: 4.8 ul

8/4/14

• Made one 250mL bottle of liquid YPD media (2%) because of contamination in other bottle
• Made sporulation media
  

  potassium acetate (1%)	2.5g
  bacto-yeast extract (0.1%)	0.25g
  glucose (0.05%)	313uL
  H2O	250mL

• Yeast roller stopped overnight, so cell density was comparatively lower than expected for today.
  

  W303A

  (1) 4.75x10^7 cells/mL -- 10.53uL for dilution

  (2) 4.05x10^7 cells/mL -- 12.35uL for dilution

  (3) 4.35x10^7 cells/mL -- 11.5uL for dilution

  1296

  (1) 4.24x10^6 cells/mL -- 118uL for dilution

  (2) 4.955x10^6 cells/mL -- 101uL for dilution

  (3) 4.15x10^6 cells/mL -- 125uL for dilution

  1294

  (1) 2.715x10^6 cells/mL -- 184uL for dilution

  (2) 2.315x10^6 cells/mL -- 216uL for dilution

  (3) 3.02x10^6 cells/mL -- 165.5uL for dilution

• The yeast was not rediluted and put back into the roller until 5:40pm.
• Made plate readings of W303A(1) for OD600

  OD600	cell density (cells/mL)
  1.556	2x10^8
  1.037	1x10^8
  0.795	5.17x10^7
  0.618	4.78x10^7
  0.417	2.51x10^7
  0.236	1.535x10^7
  0.134	8.35x10^5
  0.125	1.05x10^5

• Colony PCR'ed EST2, Mak31, Vps75 for biobricking

For tomorrow: start new yeast experiment(?), continue yeast experiment (5:40 check), autoclave more glass culture tubes, biobrick parts

8/6/14

• Gave presentation on project to summer STEM participants.
• Autoclaved culture tubes
• Checked roller at ~4:15

  W303A

  (1) 6.20x10^7 cells/mL -- 8.06uL for dilution

  (2) 5.70x10^7 cells/mL -- 8.77uL for dilution

  (3) 7.03x10^7 cells/mL -- 7.1uL for dilution

  1296

  (1) 1.47x10^7 cells/mL -- 34.0uL for dilution

  (2) 1.99x10^7 cells/mL -- 25.1uL for dilution

  (3) 1.74x10^7 cells/mL -- 28.7uL for dilution

  1294

  (1) 2.33x10^7 cells/mL -- 21.5uL for dilution

  (2) 2.41x10^7 cells/mL -- 20.7uL for dilution

  (3) 2.33x10^7 cells/mL -- 21.4uL for dilution

• Put rollers back in for one more day(to complete 7 days)
• Put 1305 diploid into sporulation media and restruck two plates of 1305.

Tomorrow: Check rollers at 4:30. PCR biobrick parts. prepare psb1c3 vector for biobricking.

8/7/14

• PCR of Biobrick Parts: VPS75::TRP1 (colony 6), MAK31:: TRP1 (colony B), EST1 (13000)
  1-2kb program with extension time 60sec and annealing temperature 56 C
  Want 500 ng of the vector: psB1C3: 71.1 ng/ul=>7ul
  

  Biobrick Digest Procedure

  ECORI Enzyme: 1ul

  Pst1 Enzyme: 1ul, should be added last.

  1 ul of enzyme: 10 ul total-> make 20 total but professor suggested 15 ul total

  1.5ul BSA, 1.5 ul BuffH, 3ul water =>15 ul total

  37 C incubation for 1 hour, 70C innactivation period 15 min.

• Purify using PCR product procedure
• Nanodrop and run on gel (expected size of vector: 2040 bp)

  Final concentration of psB1C3: 9.0 ng/uL

• Gel Run of PCR'd biobrick parts

Wells: 1 blank ; 2 Versa Ladder ; 3 EST2 ; 4 MAK31 ; 5 VPS75

Results

faint band on 1K for VPS75. Redyed but did not make clearer

• Put in two samples of 1305 in sporulation media at ~2:30 pm
• Gel Run of purified psB1c3 vector

  Wells: 1 blank ; 2 Versa Ladder ; 3 psB1c3 purified

  Results

  OD600 of yeast cells

  W303A: 1: 1.161; 2:1.143; 3:1.367

  1294: 1:1.027; 2.1.080; 3:1.079

  1296: 1: 0.987; 2.1.224; 3:1.091

For tomorrow: purify VPS75 biobrick part; double digest VPS75; insert VPS75 part into psB1c3 vector; check cells in sporulation media ; rerun PCR of EST2 and MAK31 with different settings?

8/8/14

• Redo of Colony PC EST2 and MAK31

  Used Q5 Master Mix with buffers

  12.5ul of 1X Mix

  1.25ul of FWD and RVS Primers

  10 ul of template

  25ul reaction total

• PCR Settings

  Denaturation: 98C, 30sec

  30Cycles: 98C, 30sec

  55C, 30sec

  72C, 30sec

  Extension Time: 72C, 2min

  Hold at 4C

• Gel Run of EST2 and MAK31

  Well1: blank, 2: Versa Ladder, 3: MAK31, 4: EST2

  Results

  no bands so PCR did not work

• Nanodrop malfunction
  concentration of purified VPS75 unchecked:
  double digest VPS75 and insertion of VPS75 part into psB1c3 vector not operated
• Made graph of yeast growth curve (with changed OD600 equation)

For monday: New yeast cycle, check Spo, double digest VPS75; insert VPS75 part into psB1c3 vector;

8/11/14

• Sporulation media culture

  8/6 culture contaminated

  8/7 cultured for 4 days showed clustering

  1000x KANMX selection

  2.25 ml sterile PBS

  G418 disulfate salt

  filter sterilized and refrigerated

• 1305 selection plates: RAD52:LEU2//EST2:KANMX//SGS1:HIS3
• Haploid seletion
• zymolyase breaks down membrane==>cultured on YPD plates. then replicated onto LEU-, KANMX, HIS3-
• double selection on LEU, KANMX plates
• 200ul zymolyase storage buffer to zymolyase 1000U
• VPS75 purification check: 12.2 ng/ul
• Made 3 plates each of W303A/alpha with 35 cells/mL W303A/alpha 350 cells/mL

Tomorrow: double digest VPS75 insert, biobrick transformation, replicate spo plates, screen EST1+VPS75/MAK31 colonies for double knockouts.

For future: generate growth curve for double selected spo plates (EST2+RAD52) and EST1+VPS/MAK

8/12/14

• double digested VPS75 insert

  45 uL VPS75 (12.2 ng.uL)

  1.5 uL each ECOR1 and PST1

  6 uL buffer H

  6 uL 10x BSA

  60 uL total

  Incubated for 1 hour (no heat inactivation)

• Purified VPS75 insert (Final concentration: 1.1 ng/uL)
  Need to redo PCR. Next time PCR, dilute to 50 uL, then directly double digest
• Inoculated 1296, W303alpha, VPS75::TRP1 colony 6, MAK31::TRP1 colony B for new curves. Put in roller at 2:45 pm
• colony PCR screened for double knockouts (EST1 and VPS/MAK)

  A = VPS75::TRP1 ; EST1::LEU2 "Colony 6" Plate 2 (strain 1880)

  B = MAK31::TRP1 ; EST1::LEU2 "Plate B" Plate 2 (strain 1880)

  C = MAK31::TRP1 ; EST1:: LEU2 "Plate B" Plate 1 (strain 1880)

  D = MAK31::TRP1 ; EST1:: LEU2 "Colony 7" Plate 1 (strain 1880)

  E1-E5 = MAK31::TRP1 ; EST1::LEU2 "Colony 3" Plate 2 (strain 1880)

• PCR conditions:

  Initial: 95 C, 300 sec

  40 cycles

  Denature: 95 C, 30 sec

  Anneal: 56 C, 30 sec

  Extension: 72 C, 60 sec

  Final: 72 C, 420 sec

  Hold: 4 C

• Made 4 1% gels
  Ran gels of colony PCR check for EST1

  Gel 1 Wells: 1 Versa Ladder ; 2 A3 ; 3 A4 ; 4 B3 ; 5 B4 ; 6 C3 ; 7 C4

  Gel 2 Wells: 1 Versa Ladder ; 2 D3 ; 3 D4 ; 4 E1 ; 5 E2 ; 6 E3 ; 7 E4 ; 8 E5

  Gel Results

• Checked sporulated yeast cells, dissolved membrane with 10 uL zymolyase (30 minute wait)
• inoculated two tubes of 1305 in Spo media

For tomorrow: Make YPD plates, redo BioBrick of VPS75 (do not purify PCR product), look at primers of EST1 and MAK31 (BioBrick), check sporulated yeast plates (replicate if there is growth), check rollers at 2:45

8/13/14

• YPD plates made
• VPS75 colony PCR: From colony 6, 2 samples for biobricking

  Initial: 95 C, 300 sec

  30 cycles

  Denature: 95 C, 30 sec

  Anneal: 56 C, 30 sec

  Extension: 72 C, 60 sec

  Final: 72 C, 120sec

  Hold: 4 C

• use 1300 for EST2 biobrick PCR (post senescent)
• made 2 250 mL bottles for YPD plates
• Ran gel of VPS75 biobrick (~1K bp expected)

  Gel 1 Wells: 1 Blank ; 2 QuickLoad 1Kb Lab ; 3 colony 6, VPS751 ; 4 VPS75 2 ;

  Results: 1 kB bands

• PCR of EST2, MAK31 biobrick: EXT time 90 sec, Final step: 420 sec
• yeast growth curve check: OD600 readings
  

  	OD600	Cell Density	Dilution Volume
  YPD Blank	0.112
  EST2 neat	0.61	6.1*10E7	8 uL
  VPS75 neat	1.345	1.345*10E8	4 uL
  MAK31 neat	1.318	1.318*10E8	4 uL
  W303A neat	1.209	1.209*10E8	4 uL

  
  cells diluted to 1*10E5cells/ uL, triplicate samples, put in roller at 3:30PM

• Nanodrop concentrations

  VPS751 527.0 ng/uL

  VPS752 690.9 ng/uL

For tomorrow: double digest VPS75 insert
Redo PCRs for double knockout screening
Prepare for meeting
Check yeast rollers
Check sporulated yeast plates

8/14/14

• Double Digest: (done for each VPS75 1 and VPS752)

  Buffer H: 2 uL

  BSA: 2uL

  VPS75: 3.7 uL

  ddH2O: 10.3 uL

  EcoRI: 1uL

  PstI: 1uL

  Incubate at 37C for 60 min

  Heat at 70C for 15 min

• Checked concentrations, but received negative concentrations... did calculations manually:

  VPS751: 2556.33 ng/20 uL = 127.8 ng/uL

  VPS752: 1950 ng/20 uL = 97.5 ng/uL

• Stored double digested VPS751 and VPS752 at -20C till further instructions on how to ligate.
• Ran gel of yesterday's PCR to check biobrick primers

  Wells: 1, blank; 2, versa ladder; 3, E; 4, MB; 5, M7

• Made four 1% gels
• colony PCR'ed 13 samples for double knockout (Program 334)

  PCR conditions

  ext. time 30

  anneal temp 56

  final step 420

  cycles 30

• yeast growth curve check: OD600 readings

  ;	OD600	Dilution Volume
  YPD Blank	0.116
  MAK31 1	1.246	4 uL
  MAK31 2	1.22	4 uL
  MAK31 3	1.084	4.6 uL
  EST2 1	0.178	28 uL
  EST2 2	0.242	20.7 uL
  EST2 3	0.222	22.5 uL
  VPS75 1	1.219	4.1 uL
  VPS75 2	1.192	4.2 uL
  VPS75 3	1.189	4.2 uL
  W303A 1	1.128	4.4 uL
  W303A 2	1.127	4.4 uL
  W303A 3	1.037	4.8 uL

  
  put yeast cultures in roller @ 4:00PM
• Duplicated 3 plates of sporulated cells onto

  Leu-/KANMX (Rad52::Leu2 and EST2::KANMX)

  Leu- (RAD52::LEU2)

  KANMX (EST2::KANMX)

  HIS- (SGS1::HIS3)

• Updated wiki outreach page

8/15/14

• Observed 1 cell with RAD52 and EST2 knocked out as well as 1 cell with EST2 and SGS1 knocked out
• the Leu-/KANMX plate may be missing the EST2::KANMX since those plates look identical to the Leu- Plates

  Gel 1: 1, Versa Ladder; 2, A3; 3, A4; 4, B3; 5, B4; 6, C3; 7, C4; 8, E3;

  Gel 2: 1, Versa Ladder; 2, D3; 3, D5; 4, E1; 5, E2; 6, E3; 7, E4; 8, E5;

  
  The E3 in Gel 2 looked faint so we ran E3 on gel 1 as well.
• Found protocol to isolate DNA in yeast cells

  RAD52 EST2 Knockouts

  innoculate culture (5 mL for curve)

  use 1 mL of saturated culture

  mix above with 1 mL of 60% glycerol

  vortex (label clearly)

  store at -80 C

  RAD52 EST2 Growth Curve

  Take 1/2 of colony

  innoculate in 5 mL YPD

  triplicate samples tomorrow

• Make more sporulated yeast cell plates (~500 cells)

  Zymolased with 10 uL

  Made 6 plates with ~450 cells

• Yeast Growth Curves

  	OD600	Dilution
  YPD	0.146
  W303A 1	1.074	4.6 uL
  W303A 2	0.937	5.3 uL
  W303A 3	0.994	5 uL
  MAK31 1	1.035	4.8 uL
  MAK32 2	1.25	4 uL
  MAK32 3	1.25	4 uL
  VPS 1	1.184	4.2 uL
  VPS 2	1.187	4.2 uL
  VPS 3	1.044	4.8 uL
  EST2 1	0.762	6.6 uL
  EST2 2	1.321	3.8 uL
  EST2 3	0.469	10.7 uL

• Also made a RAD52 & EST2 tube and put in roller at ~5:30PM

8/16/14

Yeast Growth Curves

	OD600	Dilution
YPD	0.134
W303A 1	0.602	8uL
W303A 2	0.592	8 uL
W303A 3	0.555	8 uL
MAK31 1	1.032	5 uL
MAK32 2	0.92	5 uL
MAK32 3	1.008	5 uL
VPS 1	0.52	7 uL
VPS 2	0.675	7 uL
VPS 3	0.719	7 uL
EST2 1	0.497	7.5 uL
EST2 2	0.681	7.5 uL
EST2 3	0.582	7.5 uL
RAD52&EST2	0.836	6 uL

Dilutions

W303A: ~8uL-> 1

VPS75: ~7uL -> 3

EST2: ~7.5 uL -> 3

MAK31: ~5uL -> 1

RAD52: ~6uL

Sporulated 2 & 3 were empty and trashed

8/17/14

Yeast Growth Curves

	OD600	Dilution
YPD	0.117
W303A 1	0.67	7.5 uL
W303A 2	0.643	7.8 uL
W303A 3	0.662	7.6 uL
MAK31 1	1.184	4.2 uL
MAK32 2	1.207	4.1 uL
MAK32 3	1.171	4.3 uL
VPS 1	0.744	6.7 uL
VPS 2	0.705	7.1 uL
VPS 3	0.724	6.9 uL
EST2 1	0.706	7.1 uL
EST2 2	1.51	3.3 uL
EST2 3	1.29	3.9 uL
RAD52&EST2	0.121	6 uL
RAD52&EST2	0.116
RAD52&EST2	0.107

• Incubated another RAD52 & EST2 in 5 mL YPD
• Put in roller @ 5:30PM
• For tomorrow: freeze saturated RAD52&EST2, make 60% glycerol

8/18/14

Yeast Growth Curves

	OD600	Dilution
W303A 1	0.23	21.7
W303A 2	0.298	16.8
W303A 3	0.225	22.2
MAK31 1	1.196	4.2
MAK32 2	1.194	4.2
MAK32 3	1.212	4.1
VPS75 1	1.115	4.5
VPS75 2	1.115	5
VPS75 3	1.084	4.7
EST2 1	0.518	9.7
EST2 2	0.678	7.4
EST2 3	0.528	9.5
RAD52&EST2	0.137
RAD52&EST2	1.018	4.5
RAD52&EST2	0.498	10

• Put in roller at 5:00pm
• Wildtype culture samples were thrown out because of evidence of contamination
• Ran gel for biobrick primers. Yielded results of ~200bp when the expected result was ~1000bp
• Made triplicate samples of RAD52 EST2 inoculated yesterday (8/17). Also froze -80 C with 1 mL yeast, 1mL 60% glycerol (2 samples).

8/19/14

• Zymolased sporulated samples w/ 15 uL @ 1:05 PM - 2 tubes - followed protocol from 8/11
• Sporulated samples turned out to be contaminated, and were thrown out
• Double Digested VPS75 1 and VPS 75 2

  Incubated @ 1:13PM 37 C for 60 min

  Heated @ 2:14PM 70C for 15 min

  followed same protocol for 8/14 double digest, total volume 20 uL

• Purified VPS75 double digests (1 and 2)

  Nanodrop results

  (1): 5.6 ng/uL

  (2): 4.2 ng/uL

• Made two sporulation tubes and put in roller (Check in 5 days)

  Used 5 mL sporulation media

  1305 (1) and 1305 (2)

• Yeast Growth Curves

  	OD600	Dilution
  YPD (blank)	0.119
  MAK31 1	1.166	4.3 uL
  MAK32 2	1.19	4.2 uL
  MAK32 3	1.176	4.3 uL
  VPS75 1	1.137	4.4 uL
  VPS75 2	1.13	4.4 uL
  VPS75 3	1.206	4.1 uL
  EST2 1	1.201	4.2 uL
  EST2 2	0.57	8.8 uL
  EST2 3	0.573	8.7 uL
  RAD52&EST2	0.114
  RAD52&EST2	0.121
  RAD52&EST2	0.117
  RAD52 EST2 (set 2)	0.111
  RAD52 EST2 (2)	0.546	9.2 uL
  RAD52 EST2 (3)	0.114

• Need to start generating curves for RAD52/EST2 as well as update graphs and std. dev./error bars

8/20/14

• Double-knockout Screening: A5, A6, B5, B6, C5, C6, D5, D6, E1, E2, E3, E4, E5. Used same plates as before, choose new colonies for plates A, B, C, and D
• swirled colonies each in 200 uL ddH2O tubes
• PCR tubes: 20 uL ddH2O+colonies, 2.5 uL reverse and forward primers (EST1)rs
• Ligation notes:
• Team page wiki ideas:
• Yeast Growth Curves

  	OD600	Dilution
  YPD (blank)	0.125
  MAK31 1	1.274	3.9 uL
  MAK32 2	1.227	4.1 uL
  MAK32 3	1.259	4.0 uL
  VPS75 1	1.241	4.0 uL
  VPS75 2	1.213	4.1 uL
  VPS75 3	1.206	4.1 uL
  EST2 1	1.476	3.4 uL
  EST2 2	1.121	4.5 uL
  EST2 3	0.609	8.2 uL
  RAD52&EST2	0.107
  RAD52&EST2	0.458
  RAD52&EST2	0.109
  RAD52 EST2 (set 2)	0.129
  RAD52 EST2 (2)	0.142
  RAD52 EST2 (3)	0.143

8/21/14:

• Made two 1% gels
• Ran gels of double knockout colony PCR screenings

  Gel 1: VersaLadder, A5, A6, B5, B6, C5, C6

  Gel 2: VersaLadder, D5, D6, E1, E2, E3, E4, E5

• yeast growth curve

  	OD600	Dilution
  MAK31 1	1.147	4.4
  MAK32 2	1.158	4.3
  MAK32 3	1.187	4.2
  VPS75 1	1.15	4.3
  VPS75 2	1.152	4.3
  VPS75 3	1.161	4.3
  EST2 1	1.265	4
  EST2 2	0.651	77
  EST2 3	0.576	8.7
  RAD52&EST2	0.101
  RAD52&EST2	0.478	10.3
  RAD52&EST2	1.393	3.6
  RAD52 EST2 (set 2)	1.4
  RAD52 EST2 (2)	1.562	8.9
  RAD52 EST2 (3)	1.116

8/22/14

Yeast Growth Curve

	OD600	Dilution
YPD (blank)	0.144
MAK31 1	1.232	4.1
MAK32 2	1.19	4.2
MAK32 3	1.197	4.2
VPS75 1	1.154	4.6
VPS75 2	1.141	4.4
VPS75 3	1.153	4.3
EST2 1	1.274	3.9
EST2 2	0.656	7.6
EST2 3	0.666	7.5
RAD52&EST2	0.105
RAD52&EST2	1.21	4.1
RAD52&EST2	1.611	3.1
RAD52 EST2 (set 2)	0.185
RAD52 EST2 (2)	0.7	7.1
RAD52 EST2 (3)	0.233	21.5

8/25/14

• Dephysophorylated vector DNA
• Ligation calculator 3:1
• Not Dephosphorylated

  45 ng insert DNA - 9 uL (5.6 ng/uL)

  30 ng vector DNA - 3 uL

  10X T4 DNA ligase buffer - 2 uL

  water - 5 uL

  T4 DNA ligase - 1 uL

  Total Volume: 20 uL

• Dephosphorylated

  45 ng insert DNA - 9 uL (5.6 ng/uL)

  30 ng vector DNA - 10 uL

  10x T4 DNA ligase buffer - 2.5 uL

  water - 2.5 uL

  T4 DNA ligase - 1.25 uL

  Total Volume: 25 uL

• 15 uL zymolase w sporulated cells (protocol on 8/11/14)
• Counted cells and averaged for 444 in sample 2 and 560 in sample 1
• Made 3 plates of each sample
• RAD52/EST2 cells from the last growth curve were contaminated with bacteria
• Made 2 bottles of 250 mL YPD

1 bottle for YPD plates

LB plates:

LB - Lennox Broth - 4g

agar 1.5% - 3g (in 200 mL)

Autoclave -> don't add things when it's too hot

Add Chlor (1000x) - 200 uL​

40% Glucose

multiply 40% by mL to get grams of dextrose

used 120g dextrose and 300 mL

stirred & heated glucose in 100 mL dH20 then added more dH20 till the desired level of 300mL

• restreaked MATA and MATα for mating type checks of 1294, 1296, 1300, 1880 (VT6, MTB, MT7)

For tomorrow/things to do

if start yeast curves, start them at noon

finish up ligation

check on plates in incubator - maybe redo kan- plate w/ ypd if we do replicate yeast

double knockout screening -check other colonies/re-do

biobrick primers

yeast dna protocol - redo w/ biobrick primers

innoculate RAD52/EST2 colony at noon

put ligations in -20C in morning

8/26/14

• ligations put in -20 C
• restreaked MAK31::TRP1 colony 7
• inoculated and put in roller at noon:

  1880

  VPS75::TRP1 colony 6

  EST2 1296

  RAD52/EST2

• yeast genomic DNA extraction protocol for biobricking

  nanodrop results

  EST2 (1300) : 41/6 ng/uL

  MAK31 (MT7) : 14.4 ng/uL

  VPS75 (VT6) : 26.0 ng/uL

• ran PCR of EST2, MAK31, VPS75 for biobricking

  30 cycles

  95 C, 30 sec

  56 C, 30 sec

  72 C, 90 sec

  72 C, 120 sec

• Made 2 250 mL bottles for YPD plates
• 1 250 mL bottle for KANMX plates (250 uL G418)

For tomorrow

Run gels of biobrick inserts

if they worked, double digest, purify

transform along with VPS75

Select more sporulated cells

Check cells growing on sporulated plates

Growth curve at noon (make triplicate samples)

check mating type of EST2/RAD52, 1294, 1296, 1300, 1880 (VT6, MTB, MT7)

mate EST2 and MAK31/VPS75 for sporulation

Research

gibson assembly (isothermal) for "super plasmid"

yeast operons ( for EST2, RAD52 )

prerecombinase done in iGEM already?

biobuilder.org -> look for beta carotene yeast, possibly use as a measure to characterize growth curves, senescence vs nonsenecence

thin layer chromatography (TLC)

gas chromatography

check iGEM deadlines

8/27/14

• Zymolased (2) and (3) sporulation samples with 15 uL at 11:30 AM, wait 30 min

  (2) after diluting 2 times 1/10, counted an average of 88 cells (with 87 and 95)

  (3) after diluting 2 times 1/10, counted on average 82 cells (with 80 and 84)

• Made 2 1% gels

  Ran gel of biobricking PCR product

  Wells: 1 blank ; 2 blank ; 3 Versa Ladder ; 4 EST2 ; 5 MAK31 ; 6 VPS75

  Gel Results

  EST2 - band on 2K bp

  MAK31 - band on 600 bp

  VPS75 -band on 1K bp

  1K expected...

• checked mating types of 1880, VT6, MTB, MT7, EST2/RAD52
• Made 1 250 mL bottle for SD plates (for mating selection) (no dropout mix added

  1 bottle KAN/LEU

  1 bottle HIS

8/29/2014

• Note: 1880 is DSY strain
• 2 250 ml LEU- plates made
• VPS75 BB transformation check

  1 white colony on 1 dephosphorylcoted plate

  dense reds on not plates==> restreaked white colonies on gridded chlor- plates

• Mating type plate of EST2/RAD52, 1880, VT6, MTB,MT7

  =>MATA:1880 MTB,VT6,EST2,RAD52

  MT7 grew on both sides (possible diploid)

• replicated 1294 1296 1300 on SD selection plate
• replicated sporulated cells

  Stopped growth curve for weekend

  Cell densities in notebook

9/4/2014

• mini prepped VPS75 BB and stored in -20C (followed protocol on P14)
• prof Medvedik innoculated the VPS75 two nights ago and re-innoculated yesterday -> put in fridge yesterday for today
• nanodrop: 86.5 ng/uL

9/5/2014

For Sequencing

10 uL plasmid in each PCR tube (2 tubes)

5 uL 5 mM FWD primer into 1 tube (labeled SL01)

5 uL 5 mM REV primer in 2nd tube (labeled SL02)

Add up how big PSB1C3 (2K) and VPS75 (1K) - those together give a DNA size of 3K

-make at least 50uL of each primer for the 5mM