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