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
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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 sterile ddH2O or PBS to make 10ul of cell solution
-
Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube
+
'''If screening for successful clones, 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)
-
:Note: Program can be optimized by shortening to 21 cycles.
+
:Note: Program can be optimized, shortening to 21, or slightly fewer, 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, though it requires increasing ddH2O to 50-200 uls (diluting until mostly translucent). This allows for screening of entire plate with a single PCR reaction. This can be very useful when transformations are not much higher than background vector-only controls. It allows for excluding the possibility of missing a single positive colony on a plate. Of course, individual colonies or regions would then have to be re-screened, but this can require less time than a complete reconstruction process in many circumstances.

Latest revision as of 03:02, 29 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 successful clones, 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, or slightly fewer, cycles.
Multiple colonies can be screened in the same reaction for insert if using insert-specific primers, though it requires increasing ddH2O to 50-200 uls (diluting until mostly translucent). This allows for screening of entire plate with a single PCR reaction. This can be very useful when transformations are not much higher than background vector-only controls. It allows for excluding the possibility of missing a single positive colony on a plate. Of course, individual colonies or regions would then have to be re-screened, but this can require less time than a complete reconstruction process in many circumstances.
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