SBB12Ntbk-Tina Nie: Difference between revisions
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==--[[User:Tina Nie|Tina Nie]] 14:25, 16 February 2012 (EST))== | ==--[[User:Tina Nie|Tina Nie]] 14:25, 16 February 2012 (EST))== | ||
First day of labwork: Set up two PCR reactions. | First day of labwork: Set up two PCR reactions. | ||
Set up wobble reaction for: | Set up wobble reaction for sbb1227: | ||
{ | <pre> | ||
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 | |||
</pre> | |||
Using protocol: | Using protocol: | ||
Line 12: | Line 24: | ||
5 uL Oligo 2 (100uM) | 5 uL Oligo 2 (100uM) | ||
0.75 uL Expand Polymerase 1 | 0.75 uL Expand Polymerase 1 | ||
Set up PCA1 reaction for sbb1213: | |||
<pre> | |||
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 | |||
</pre> | |||
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. |
Revision as of 12:29, 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.