SBB12Ntbk-Karen D. Alvarez

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Karen D. Alvarez 14:43, 16 February 2012 (EST)

PCR for part sbb1217

1. ca998/osbb1333R on template pBca9145-jtk2768

 a)osbb1333R oligo is 34.6nM, MW=11,944.7, Tm=72.1°C. I prepared a dilute solution of 10μM.
 b) I mixed the following:

24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo ca998, 10uM 1uL Oligo osbb1333R, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA

2. osbb1333F/g00101 on template pBca9525-Bca1832

 a)osbb1333F oligo is 34.0nM, MW=6,766.4, Tm=60.4°C. I prepared a dilute solution of 10μM.
 b) I mixed the following:

24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo g00101, 10uM 1uL Oligo osbb1333F, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA

3. Place oligos in ice bucket for PCR program according to predicted PCR product base pair length.

 a)ca998/osbb1333R PCR product is 643 bp
 b)osbb1333F/g00101 PCR product is 1438 bp

==PCA for sbb1206 part (leucine part) 1. PCA1 on o1,o6,o2 (pca1) 2.I mixed 1μL of each oligo 3. I followed the instructions below: 38 uL ddH2O 5 ul 10x expand buffer 5 ul 2mM dNTPs 1 ul oligo mixture ( 0.75 ul Expand polymerase