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Team:Washington/Protocols/Colony
From 2011.igem.org
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=Colony PCR with Green tag= | =Colony PCR with Green tag= | ||
| - | Master mix(7ul): | + | Master mix (7ul): |
1ul 10uM forward primer | 1ul 10uM forward primer | ||
| Line 12: | Line 12: | ||
5ul 2x Green tag | 5ul 2x Green tag | ||
| - | Cell water(3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water | + | Cell water (3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water |
Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube | Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube | ||
Use program "Colony" & change the extension time (1 min per kb) | Use program "Colony" & change the extension time (1 min per kb) | ||
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| + | ::Note: Program can be optimized shortened to 21 cycles. | ||
| + | ::Also, multiple colonies can be screened in the same reaction for insert if using insert-specific primers, while increasing ddH2O to 50-200 uls (Dilute until mostly translucent). This allows for screening of theoretically and entire plate if the ratio of transformation is not much over background, to exclude the possibility of missing single colonies. | ||
Revision as of 01:50, 14 September 2011
Colony PCR with Green tag
Master mix (7ul):
1ul 10uM forward primer
1ul 10uM reverse Primer
5ul 2x Green tag
Cell water (3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water
Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube
Use program "Colony" & change the extension time (1 min per kb)
- Note: Program can be optimized shortened to 21 cycles.
- Also, multiple colonies can be screened in the same reaction for insert if using insert-specific primers, while increasing ddH2O to 50-200 uls (Dilute until mostly translucent). This allows for screening of theoretically and entire plate if the ratio of transformation is not much over background, to exclude the possibility of missing single colonies.
