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Yeast Vector Library
7/1/11
* Made growth plates for the 12 vectors * 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
