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

Annie Joseph 12:30, 21 February 2012 (EST)

For the sbb1216 part, I ran the two PCR products from last week on the agarose gel for gel purification: PCR ca998/osbb1333R on pBca9145-jtk2768 (A) PCR osbb1216F/g00101 on pBjk2741-jtk3346 (B) Obtained alright results, can be seen on the gel two lanes 8 and 9. Cut out the bands and did the rest of the gel purification for both A and B. For the Leucine Zipper part (sbb1205): I had the pca2 product from last week, which I ran on gel for analysis (lane 4 ?) looks alright. Did a cleanup of the pca2 product (did the protocol for regular zymo cleanup which I think might have been better to use the small frag cleanup given the size of the pca2) and proceeded to do the digest of pca2 with NheI/BamHI following the protocol for EcoRI/BamHI Digest of Wobble Products for the size considerations. Kept the digest on 37 degrees on the thermocycler for ~1 hour. Finally did a zymo small fragment cleanup this time.