SBB11Ntbk-Gary Dixon

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 between both 53 oligos. Placed in <2kb PCR because PCR product should be 981.

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