SBB12Ntbk-Annie Joseph

Annie Joseph 16:23, 13 February 2012 (EST)

We did initial PCR/PCA today!

For part sbb1216:

Obtained and made a 10 micro molar solution of oligos ca998/osbb1333R and osbb1216F/g00101 for PCR.

Set up two solutions for PCR:

This one I call A in construction file:

24uL ddH2O

3.3uL 10x Expand Buffer "2"

3.3uL dNTPs (2mM in each)

1uL ca998, 10uM

1uL osbb1333R, 10uM

0.5uL Expand polymerase "1"

0.5uL pBca9145-jtk2768

And...

This one is B:

24uL ddH2O

3.3uL 10x Expand Buffer "2"

3.3uL dNTPs (2mM in each)

1uL osbb1216F, 10uM

1uL g00101, 10uM

0.5uL Expand polymerase "1"

0.5uL pBjk2741-jtk3346

For part sbb1205

I set up a solution based on the PCA protocol on open wet ware.

38 uL ddH2O

5 ul 10x expand buffer

5 ul 2mM dNTPs

1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)

0.75 ul Expand polymerase

Notes:

n/a

Annie Joseph 12:30, 17 February 2012 (EST)

Ran two lanes on agarose gel for gel purification of products A and B from last time. Extracted the product A from the gel. The lane for B was very faint so I have decided to redo the gel one more time before considering whether it was a mistake in an earlier step :(

Created the PCA solution for creation of pca2.

For next time: I will rerun the gels (PCR A&B with loading buffer), extract Create the digests for the part and template and use thermocycler (time for ligation ?)

Possibly with time remaining, I will set up the pcr reaction with A+B