SBB12Ntbk-Tina Nie

--Tina Nie 14:25, 16 February 2012 (EST))

First day of labwork: Set up two PCR reactions. Set up wobble reaction for sbb1227:

Wobble tn001F/tn002R            (64bp, wobpdt)
Digest wobpdt                   (NheI/BamHI, L, wobdig)
Digest pBca9525-Bca1834         (NheI/BamHI, L, vectdig)
Ligate wobdig and vectdig (pBca9525-sbb1227) {Leu8}
----------------------------------------------------------------------
>tn001F Forward construction of sbb1227 basic part
ccataGCTAGCggcagtggatctggtCTGCTACTGTTGCTG
>tn002R Reverse construction of sbb1227 basic part
CTGATggatccTTACAGGAGTAGCAGCAACAGTAGCAGaccag
>wobpdt
ccataGCTAGCggcagtggatctggtCTGCTACTGTTGCTGCTACTCCTGTAAggatccATCAG

Using protocol:

 29 uL water
 5 uL Expand 10x Buffer 2
 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
 5 uL Oligo 1 (100uM)
 5 uL Oligo 2 (100uM)
 0.75 uL Expand Polymerase 1

Set up PCA1 reaction for sbb1213:

PCA1 on o15,o11,o12       (pca1)
PCA2 with o15/o12 on pca1 (139 bp, pca2)
Digest pca2               (NheI/BamHI, L, 1213dig)
Digest pBca9525-Bca1834   (NheI/BamHI, L, vectdig)
Ligate 1213dig + vectdig, product is pBca9525-sbb1213
----
>o15	
CCATAgctagcGGCAGTGGATCTGTTAAAGAAGCGGAAGACAAAAACGAAGAACTGCTGAGT
>o11
CAAAAACGAAGAACTGCTGAGTGCCGCTTACCACGCAGCCAACGAAGTTGCTCGTCTGA
>o12
CAGTAGGATCCTTAGCCGCCACGTTCGCCAACCAGTTTTTTCAGACGAGCAACTTCGTT
>pca2
CCATAgctagcGGCAGTGGATCTGTTAAAGAAGCGGAAGACAAAAACGAAGAACTGCTGAGTGCCGCTTACCACGCAGCCAACGAAGTTGCTCGTCTGAAAAAACTGGTTGGCGAACGTGGCGGCTAAGGATCCTACTG

Using protocol:

1. 38 uL ddH2O
2. 5 ul 10x expand buffer
3. 5 ul 2mM dNTPs
4. 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
5. 0.75 ul Expand polymerase

Both programs run for us after class.

Dilutions used: