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Yeast Vector Library
7/1/11
- Made growth plates for the 12 vectors
-pRS 403: HIS3 Selectable, Integrating -pRS 404: TRP1 Selectable, Integrating -pRS 405: LEU2 Selectable, Integrating -pRS 406: URA3 Selectable, Integrating -pRS 413: HIS3 Selectable, CEN/ARS Episomal -pRS 414: TRP1 Selectable, CEN/ARS Episomal -pRS 415: LEU2 Selectable, CEN/ARS Episomal -pRS 416: URA3 Selectable, CEN/ARS Episomal -pRS 423: HIS3 Selectable, 2micron Episomal -pRS 424: TRP1 Selectable, 2micron Episomal -pRS 425: LEU2 Selectable, 2micron Episomal -pRS 426: URA3 Selectable, 2micron Episomal
- Repipetted primers
- Started PCR experiment design
7/2/11
- Created 2 mL overnight cultures of vectors in E. coli w/ LB + Carbenicillin
- 37C incubation
7/3/11
- Miniprep 12 vectors from 2 mL overnights
- Store in -20C
7/5/11
- Designed the multiple cloning site substitution and QuikChange protocol to run by Dr. Boeke
7/6/11
- Created and consolidated all PCR reagents and DNA for tomorrow's PCR
7/7/11
- Multiple cloning site substitution PCR
7/8/11
- DpnI Digest
- Ligation
7/11/11
- Transformation and plating
7/12/11
- No colonies
- Repeat transformation and plating
7/13/11
- No colonies
- Optimized PCR protocol according to Agilent's Herculase manual
7/14/11
- Regrew original pRS vectors in overnight cultures
7/15/11
- Mini prep vectors
- MCS delete/insert PCR
7/16/11
- PCR Purification
7/18/11
- DpnI digest
- Overnight ligation at 16C
7/19/11
- Transformation of 12 vectors
7/20/11
- No colonies
- Verify original vectors with EcoRI digest
- Verification successful
7/21/11
- Re-do transformation
7/22/11
- Transformation failed
- Change QCPCR protocol to new two-step process
- Create competent cells
7/25/11
- Try new Site Directed Mutagenesis QCPCR protocol on pRS 416 @ various template and primer concentrations
7/26/11
- Yeast spec. assay
- DpnI digest
- 416 Site Directed Mutagenesis Transformation
7/27/11
- Bad competent cells (no colonies on pos. ctrl.)
- Redo QCPCR w/ 9 primer/template concentrations
7/28/11
- DpnI digest of 416 QCPCR
- Transformation using high efficiency TOP10 cells
7/29/11
- 416 SDM QCPCR worked!
- Grow overnight cultures
8/1/11
- Miniprep
- 2nd step QCPCR
8/2/11
- DpnI digest
- Transformation
8/3/11
- Transformation successful: finished 416 biobricked vector
- Grow overnight culture
8/4/11
- Create new competent cells using TSS protocol
8/25/11
- 1st QCPCR on rest of vectors
- DpnI digest
- Transformation
8/26/11
- Colonies for all but 413 and 403
- Grow overnight cultures
8/27/11
- Miniprep successful transformations
8/29/11
- 2nd QCPCR on successful 1st QCPCRs
8/30/11
- DpnI digest
8/31/11
- QCPCR (site directed mutagenesis) on 403, 405, 413, 415, 423
9/1/11
- Digest EcoRI, XbaI, SpeI and PstI on final biobricked vector (416)
- Run Gel: Failed (PstI had 3 products instead of 1)
9/5/11
- DpnI digest 8/31 reactions
- Transform 8/31 reactions
9/6/11
- Colonies for 403, 405, 413, 415, 423, 424, 425
9/7/11
- Redo first second round QCPCR (multiple cloning site swap) 9/6 colonies
9/8/11
- PCR failed: Samples evaporated in PCR machine
9/12/11
- Redo first round (site directed mutagenesis) on all pRS vectors
9/13/11
- DpnI digest
- Transform 9/7 QCPCR
9/14/11
- No colonies
- Problem may be in incompatible PCR buffer (Herculase buffer) and Transformation buffer. Solution is to do PCR purification before transformation.
- Check theory by running PCR purification on PCR products that previously failed transformation and re-transform
9/15/11
- Colonies! Theory confirmed.
- Re-run all QCPCR (site directed mutagenesis) reactions
9/16/11
- DpnI digest
- Transform
9/17/11
- All QCPCR reactions had large amount of colonies
- Miniprep all samples
9/20/11
- Restriction enzyme digest check on all miniprepped vectors
