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Team:Washington/Protocols/Colony
From 2011.igem.org
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(→Colony PCR with Green tag) |
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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 | + | Cell water (3ul): Pick one colony from the plate and mix with 10ul of sterile ddH2O or PBS to make 10ul of cell solution |
| - | Reaction = Master mix(7ul) + Cell | + | '''If screening for positive results, the cell solution should be retained''' |
| + | |||
| + | '''Sterile glycerol can be added to 50% final concentration, and cell solution can be frozen at -80<sup>o</sup>C for use at a later date''' | ||
| + | |||
| + | Reaction = Master mix(7ul) + Cell solution (3ul) = 10ul total per tube | ||
Use program "Colony" & change the extension time (1 min per kb) | Use program "Colony" & change the extension time (1 min per kb) | ||
| Line 20: | Line 24: | ||
:Note: Program can be optimized, shortening to 21 cycles. | :Note: Program can be optimized, shortening to 21 cycles. | ||
: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 positive colonies. | :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 positive colonies. | ||
| + | (QUESTION: HOW DOES DECONVOLUTION WORK IN THE ABOVE INSTANCE? IS THIS JUST TO REAMPLIFY INSERT?) | ||
Revision as of 02:49, 16 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 sterile ddH2O or PBS to make 10ul of cell solution
If screening for positive results, the cell solution should be retained
Sterile glycerol can be added to 50% final concentration, and cell solution can be frozen at -80oC for use at a later date
Reaction = Master mix(7ul) + Cell solution (3ul) = 10ul total per tube
Use program "Colony" & change the extension time (1 min per kb)
- Note: Program can be optimized, shortening to 21 cycles.
- 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 positive colonies.
(QUESTION: HOW DOES DECONVOLUTION WORK IN THE ABOVE INSTANCE? IS THIS JUST TO REAMPLIFY INSERT?)
