Plasmid Purification and Sequencing; Start of Leu Zipper Peptide Gateway Reaction, 5 March 2009
1) Pelleted ~3mL of liquid culture using 2 rounds of 30sec. spins. 2) Added 250uL P1 buffer (fridge) to dry cells. Resuspended with vortexer. 3) Added 250uL P2 lysis buffer and mixed until clearer. 4) Added 350uL N3 buffer to cause debris precipitation. Shook for 1min. Spun down for 5min. 5) Decanted remaining liquid into blue miniprep columns. Spun at 12k rpm for 30sec. 6) Washed with 500uL PB buffer. Spun 15sec. 7) Washed with 750uL PE buffer. Spun 15sec. 8) Spun an extra 60sec. to dry. 9) Eluted DNA in new tubes using 50uL ddH20. Spun 30sec at 14k rpm. 10) Aliquoted 30uL purified plasmid for sequencing.
Ligation and Transformation, 2 March 2009
Performed a ligation of the 4K55 PCR product without gel purification with pBca9495AK predigested vector and plated on Amp (single red line marking). Product is pBca4945AK-m10048. Used the remaining 7uL to gel purify. Kept DNA-agarose-ADB solution in drawer.
Ligation of EcoRI/BamHI digest
1) Combined 6.5uL ddH2O, 1uL T4 ligase buffer, 1uL vector digest, 1uL insert digest, and 0.5uL T4 ligase in small tube. Reagents located on ice block. 2) Mixed and incubated at RT for 30min. 3) Put on ice.
Transformation by heat-shock
1) Added 75uL of competent cells to to ligation mixture. 2) Incubated at 0C for 10min and heat-shocked at 42C on heating block for 2min. 3) Let cool on ice, and plated entire volume immediately. 4) If marker is not Amp, add 100uL LB broth, put in the 37C shaker for 40min., and then plate.
PCR Analysis, 27 February 2009
Found that the 4K60 PCR protocol did not work on the INP repeats - the analytical gel showed no bands. Still ran a digest and zymo cleanup. Discarded the two tubes (duplicates) on 03/04 after finding colony growth from workup of the first 4K55 reaction.
Alternate PCR run; Digest and Cleanup, 25 February 2009
Found significant streaking during gel purification (which need not be performed prior to ligation for vanilla PCR) of INP fragment. Performed two more PCR reactions with a higher melting temperature. Performed a regular Zymo cleanup to continue working up the first run.
1) Transferred 8uL of PCR product into a PCR tube. 2) Added 1uL of NEB buffer 2 and 0.5uL of BamHI/EcoRI, all located on the freezing block. 3) Incubated at 37C on thermocycler for 45min. 4) Started gel purification by visualizing upstairs. Note: Gel had insufficient Sybr dye.
PCR Analysis and Cleanup, 23 February 2009
Ran an analytical gel and performed the regular zymo cleanup.
To do for Wednesday: Perform the EcoRI/BamHI digest, run a purification gel, perform a second zymo cleanup, ligate into the vector, and transform by heat shock.
1) Prepared loading solution with 5uL PCR product and 5uL dilute loading buffer. 2) Gel ran for 30 min until ladders were resolved. 3) Verified 4kB band in lane 8 by comparison with Fermentas Gene Ruler 1kB DNA Ladder Plus.
Regular Zymo Cleanup
1) Added remaining PCR reaction (27uL) to 180uL Zymo ADB buffer (brown bottle) in Zymo column (clear color). 2) Spun at 14k rpm for 30sec. Discarded waste. 3) Added 200uL Zymo wash buffer (70% EtOH) and spun at 14k rpm for 30sec. Discarded waste. Performed step twice. 4) Spun an extra 90 sec. to dry. 5) Eluted with 27uL ddH2O into clean eppendorf.
PCR, 23 February 2009
Summary for 18 February 2009: received and reconstituted the Oeh001F/Oeh002R oligos and performed PCR.
1) Performed quick spin on tubes to pellet the DNA powder. 2) Added 288uL ddH2O to 288nmol of oligos to reach a final concentration of 100uM. 3) Took out a 1uL aliquot and diluted ten-fold to 10uM for PCR recipe.
1) Combined the following: 24uL ddH2O, 3.3uL 10x buffer "2", 3.3uL dNTPs (2mM), 1uL FP/RP (10uM), 0.5uL of pBca1256-Bca1346 DNA. 2) Added 0.5uL Expand Polymerase "1". 3) Used the C4K55 thermocycler protocol for an expected product ~4kb.