SBB11Ntbk-Gary Dixon

Gary Dixon 15:13, 22 February 2011 (EST)

Used the wrong Wash buffer for zymo cleanups, so Professor Anderson redid the PCRs.

Ran another analytical gel for SOEing product sbb1127.

Performed another Zymo cleanup on sbb1127. Did not start digestion because running low on time.

EcoRI/BamHI digest of Part 1117: http://openwetware.org/wiki/Template:SBB-Protocols_Enz2

Entered thermocycler at 10:24. Removed for Prep gel at 11:25.

Prep gel started at 11:43.

Cut out after class by Professor Anderson. Thanks Professor!

Gary Dixon 16:36, 18 February 2011 (EST)

Ran an analytical gel for the SOEing PCR product that used 53/53 oligos.

Gel confirmed that the major PCR product is around 981bp. There is another band that is not the desired product. It looks like it is around 300bp, which suggests that is a result of the B template from the first PCR reaction. There will be a round of gel purification after digestion, so they will be separated.

Proceeded to clean up the PCR product using the Regular Zymo Cleanup Protocol: http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1

Put into box A labeled as GD 53/53 PCRP

Gary Dixon 13:22, 17 February 2011 (EST)

Running two preparative gels:

53/31 PCR = GD1 (#3)

32/53 PCR = GD2 (#4)

5ul of product

5ul of loading dye

And one analytical gel:

42/42= GD3 (#6)

2ul of product

5ul of loading dye

Both gels looked good.

Ran preparative gel, did surgery, and melted the gel with 600ul of ADB buffer.

Did regular Zymo clean-up for 42/42. Storing it in box as GD 42/42 PCRP.

Did Zymo gel purification for 32/53 combined with 53/31.

Used the product of the Zymo gel purification as the template for the PCR with both 53 oligos. Placed in <2kb PCR because PCR product should be 981bp.

Gary Dixon 14:32, 15 February 2011 (EST)

Performing 3 PCR reactions. One for part sbb1117, and two for part sbb1127 (soeing).

First, sorted out some confusion about letter R and F on oligo. R and F don't necessarily correspond to your forward and reverse oligo. So ss53r is the forward oligo for P_nmpC and ss53f is the reverse.

Then diluted oligos gd031 and gd032.

Then went through the standard procedure to set up PCR reactions. I have three in total.

ss53r/gd031R (718bp, gp = A)

gd032F/ss53f (287bp, gp = B)

ss42r/ss42f (1032bp)

The procedure: http://openwetware.org/wiki/Template:SBB-Protocols_PCR2

Put all three PCR reactions in the <2,000kb reaction.

Gary Dixon 23:59, 5 February 2011 (EST)

Hello World