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): W303α+TRP1-1, W303α negative control, W303α+TRP1-3
• Photos of yeast plates/restreaked: 1300, 1294, 1296,1297, w303α
  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°C instead of 54°C.

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 photos
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, W303α, 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°C->56&degC, maybe try 58°C)




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 W303α, 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/μL

  LEU2: 163.8 ng/μL

• 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°C, 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°C, Time 300

  Anneal: Temp 56°C, Time 30

  Extend: Temp 72°C, Time 30

  Final Step: Temp 72°C, Time 420

  Final Hold: Temp 4°C

  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 500μL 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+250μL ethidium bromide. After staining, confirmed that 2 colonies had knockouts (3 and 6)
• Streaked out three plates of previous W303α 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°C, Time 300

  Denaturing: Temp 95°C, Time 30

  Anneal: Temp 54°C, Time 30

  Extend: Temp 72°C, Time 120

  Final Step: Temp 72°C, Time 420

  Final Hold: Temp 4°C

  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/μL

  LEU: 122.2 ng/μL

• 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/μL

  EL2: 4.8 ng/μL

  EL3: 2.7 ng/μL

  EL4: 13.6 ng/μL

• 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 W303α 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	2.5μL	22.5μL
  Primers (Forward & Reverse)	5μL	45μL
  10mM dNTP	0.25μL	2.25μL
  Template	5μL
  Taq DNA Polymerase	0.5μL	4.5μL
  ddH2O	11.75μL	105.75μL
  Total	25μL	180μL

• 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