Endy:Colony PCR protocol
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Solutions/reagents:
- Thermo polymerase buffer
- dNTPS
- Forward primer
- Reverse primer
- Taq or Vent Polymerase
- Template suspension
- H2O
Equipment:
- Thermocycler
- Reaction tubes
Steps:
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water.
- Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.
- Reaction mix
Set up a reaction in reaction tube (1) as follows on ice:
<thead></thead><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body>Initial concentration Final concentration Final volume per 10 µl reaction Thermo polymerase buffer 10X 1X 1 µl dNTPS 10X 1X 1 µl Forward primer 40 µM 0.6 µM 0.15 µl Reverse primer 40 µM 0.6 µM 0.15 µl Taq or Vent Polymerase -- -- 0.1 µl Template suspension -- -- 6.6 µl H2O -- -- 1 µl - PCR protocol
Program a standard thermocycler to run the reaction using the following parameters:
Initial denaturation- Denature: 95°C, 6 mins
- No. of cycles: 35
- Denature: 95°C, 30 secs
- Anneal: 53°C, 30 secs
- Elongate: 72°C, 60 secs
- Elongate: 72°C, 10 mins
- Hold: 4°C, until removed from machine
For shorter amplicons, under ~1kb, this can be shortened judiciously.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 28 mins
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