SBB09Ntbk-Eric Hsieh

Eric W. Hsieh 14:11, 25 February 2009 (EST)

EcoRI/BamHI digest

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.

Eric W. Hsieh 14:39, 23 February 2009 (EST)

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.

Analytical Gel

1) Prepared loading solution with 5uL PCR product and 5uL dilute loading buffer.
2) Gel ran for x 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. 

Eric W. Hsieh 11:22, 23 February 2009 (EST)

Summary for 18 February 2009: received and reconstituted the Oeh001F/Oeh002R oligos and performed PCR.

Oligos

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.

PCR

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. 

Eric W. Hsieh 12:07, 11 February 2009 (EST)

1) Created the construction file for the Ice Nucleation protein repeats.

2) Created this notebook page and added a profile picture.