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User:Bipedalape/1 July 2008
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<br>PCR: MioC (Promoter Region from JM109) | <br>PCR: MioC (Promoter Region from JM109) | ||
| - | Primers | + | Primers: |
Reverse - Site Directed Mutation to Produce Spe1 Restriction Site | Reverse - Site Directed Mutation to Produce Spe1 Restriction Site | ||
| + | |||
5' AAC TGC AGC GGC CGC TAC TAG TAA TTG TAC GCT GTG AAC GC 3' | 5' AAC TGC AGC GGC CGC TAC TAG TAA TTG TAC GCT GTG AAC GC 3' | ||
Forward - | Forward - | ||
| + | |||
5' CGG AAT TCG CGG CCG CTT CTA GAG GCC AAA GAC TAC CCT TCC 3' | 5' CGG AAT TCG CGG CCG CTT CTA GAG GCC AAA GAC TAC CCT TCC 3' | ||
PCR settings: | PCR settings: | ||
| + | |||
95 3min | 95 3min | ||
| + | |||
{95 0.5 min | {95 0.5 min | ||
| + | |||
50 0.5 min | 50 0.5 min | ||
| + | |||
72 1 min) | 72 1 min) | ||
| Line 18: | Line 24: | ||
72 10 min | 72 10 min | ||
| + | |||
4 hold | 4 hold | ||
Revision as of 19:38, 25 October 2008
PCR: MioC (Promoter Region from JM109)
Primers:
Reverse - Site Directed Mutation to Produce Spe1 Restriction Site
5' AAC TGC AGC GGC CGC TAC TAG TAA TTG TAC GCT GTG AAC GC 3'
Forward -
5' CGG AAT TCG CGG CCG CTT CTA GAG GCC AAA GAC TAC CCT TCC 3'
PCR settings:
95 3min
{95 0.5 min
50 0.5 min
72 1 min)
(Repeated X 30)
72 10 min
4 hold
PCR Product of ~300bp (Successful) w/o DMSO
Verification of Antibiotic Plates Potency: Colonies were isolated on Chloro, Kana, Amp, and Tet. Successful
