SBB10Ntbk-JoannaChen

Joanna Chen 18:06, 22 February 2010 (EST)

I did a Zymo cleanup on the product of the assembly reaction. The purified products are in tubes labeled "sbb17 PCA Asbly Zymo 2/22/10 JC" and "sbb18 PCA Asbly Zymo 2/22/10 JC" in Box C.

Then, I set up the amplification reaction by combining:

1. 32.5 ul ddH2O
2. 10 ul 5x phusion buffer
3. 5 ul 2mM dNTPs
4. 1 ul purified assembly reaction product
5. 1 ul each outer oligo
6. .5 ul phusion

These are in PCA tubes labeled "sbb17 PCA Ampl." and "sbb18 PCA Ampl." and placed in the ice bucket for PCA.

Joanna Chen 17:25, 17 February 2010 (EST)

Today was the first day of wet lab.

I obtained the tubes containing the oligo mixes and set up the assembly portion of PCA for both of my parts (sbb17 and sbb18). I combined:

1. 38 uL ddH2O
2. 5 ul 10x expand buffer
3. 5 ul 2mM dNTPs
4. 1 ul oligo mixture
5. 0.75 ul Expand polymerase 

PCA tubes are labeled "sbb17 PCA" and "sbb18 PCA" respectively, and placed into the ice bucket for PCA.

Last week

I designed my project parts for two similar zinc finger domains, (zf+) and (zf-), called sbb17 and sbb18 respectively. I aligned the two sequences using protein BLAST to find out where the sequences differed. Originally, I used GeneDesign on both sequences to generate oligos of target length 50bp, and then adjusted the ends of some oligos to increase the number of oligos that were the same in sbb17 and sbb18. However, this did not appear to work due to internal homology in the zinc finger domains. Later, Prof. Anderson provided me with a piece of code that generated nucleotide sequences that minimized internal repeats. I modified this code to first generate a nucleotide sequence for one of the zinc finger domains, and then generate the nucleotide sequence for the other zinc finger domain based on the sequence of the first and changing only what was necessary to produce the correct amino acid sequence. I then used GeneDesign to create oligos for these nucleotide sequences, and adjusted the ends of some oligos to increase the number of oligos that were the same in sbb17 and sbb18. In silico tests indicated that these oligos would assemble.