SBB12Ntbk-GregFukushima: Difference between revisions
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==[[User:Greg_Fukushima|Greg Fukushima]], 7 February 2012== | ==[[User:Greg_Fukushima|Greg Fukushima]], 7 February 2012== | ||
This is my first day of notebooks for 140L! I love wikis! | This is my first day of notebooks for 140L! I love wikis! | ||
| Line 5: | Line 4: | ||
==[[User:Greg_Fukushima|Greg Fukushima]], 16 February 2012== | ==[[User:Greg_Fukushima|Greg Fukushima]], 16 February 2012== | ||
First Day of PCR and cloning: | First Day of PCR and cloning: | ||
===Construction File for SOEing PCR=== | |||
<pre> | |||
PCR ca998/gfRevPR on pBjk2741-jtk2801 (160bp, A) | |||
PCR gfForRbsCmR/gfRevRbsCmR on pKGC438 (920bp, B) | |||
PCR ca998/gfRevRbsCmR on A+B (1059bp, pcrpdt) | |||
Digest pcrpdt (EcoRI/BamHI, L, pcrdig) | |||
Digest pBgl00001-Bjk2828 (EcoRI/BamHI, L, plasdig) | |||
Ligate pcrdig and plasdig, product is pBgl00001-sbb1220 | |||
---- | |||
>ca998 Forward external annealing for purification of P_R part | |||
gtatcacgaggcagaatttcag | |||
>gfForRbsCmR | |||
ggtgataatggttgcGGATCTCCACAACGGTTTCCCTC | |||
>gfRevRbsCmR | |||
gctagGGATCCTTACGCCCCGCCCTGCCAC | |||
>gfRevPR | |||
AGATCCgcaaccattatcacc | |||
>pcrpdt | |||
GTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGAAGTGGAATTCATGAGATCTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCGGATCTCCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGAGCGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACGATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCCCTAGC | |||
</pre></blockquote> | |||
Protocol for PCR: | Protocol for PCR: | ||
Made an oligo dilution of: | |||
9uL Water | 9uL Water | ||
1uL 100uM oligo | 1uL 100uM oligo | ||
Set up the following reaction in a PCR tube: | Set up the following reaction in a PCR tube: | ||
| Line 22: | Line 42: | ||
0.5uL Template DNA | 0.5uL Template DNA | ||
</pre> | </pre> | ||
Actual reaction was done by Zach. | |||
Revision as of 20:54, 18 February 2012
Greg Fukushima, 7 February 2012
This is my first day of notebooks for 140L! I love wikis!
Greg Fukushima, 16 February 2012
First Day of PCR and cloning:
Construction File for SOEing PCR
PCR ca998/gfRevPR on pBjk2741-jtk2801 (160bp, A) PCR gfForRbsCmR/gfRevRbsCmR on pKGC438 (920bp, B) PCR ca998/gfRevRbsCmR on A+B (1059bp, pcrpdt) Digest pcrpdt (EcoRI/BamHI, L, pcrdig) Digest pBgl00001-Bjk2828 (EcoRI/BamHI, L, plasdig) Ligate pcrdig and plasdig, product is pBgl00001-sbb1220 ---- >ca998 Forward external annealing for purification of P_R part gtatcacgaggcagaatttcag >gfForRbsCmR ggtgataatggttgcGGATCTCCACAACGGTTTCCCTC >gfRevRbsCmR gctagGGATCCTTACGCCCCGCCCTGCCAC >gfRevPR AGATCCgcaaccattatcacc >pcrpdt GTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGAAGTGGAATTCATGAGATCTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCGGATCTCCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGAGCGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACGATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCCCTAGC
Protocol for PCR:
Made an oligo dilution of:
9uL Water 1uL 100uM oligo
Set up the following reaction in a PCR tube:
24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo 1, 10uM 1uL Oligo 2, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA
Actual reaction was done by Zach.
