SBB11Ntbk-Gary Dixon: Difference between revisions
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Did Zymo gel purification for 32/53 combined with 53/31. | 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 | 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. | ||
==[[User:Gary Dixon|Gary Dixon]] 14:32, 15 February 2011 (EST)== | ==[[User:Gary Dixon|Gary Dixon]] 14:32, 15 February 2011 (EST)== |
Revision as of 13:36, 17 February 2011
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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