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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
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
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
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
