SBB12Ntbk-Tina Nie: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 44: | Line 44: | ||
Using protocol: | Using protocol: | ||
38 uL ddH2O | # 38 uL ddH2O | ||
5 ul 10x expand buffer | # 5 ul 10x expand buffer | ||
5 ul 2mM dNTPs | # 5 ul 2mM dNTPs | ||
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) | # 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) | ||
0.75 ul Expand polymerase | # 0.75 ul Expand polymerase | ||
Both programs run for us after class. | Both programs run for us after class. |
Revision as of 12:30, 16 February 2012
--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:
- 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
Both programs run for us after class.