Endy:Colony PCR protocol

From OpenWetWare

Solutions/reagents:

• Thermo polymerase buffer
• dNTPS
• Forward primer
• Reverse primer
• Taq or Vent Polymerase
• Template suspension
• H₂O

Equipment:

• Thermocycler
• Reaction tubes

Steps:

1. Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water.
   
2. 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.
   
3. Reaction mix
   Set up a reaction in reaction tube (1) as follows on ice:
   
   

   	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

4. PCR protocol
   Program a standard thermocycler to run the reaction using the following parameters:
   Initial denaturation
   
   • Denature: 95°C, 6 mins
   Thermocycling
   
   • No. of cycles: 35
   • Denature: 95°C, 30 secs
   • Anneal: 53°C, 30 secs
   • Elongate: 72°C, 60 secs
   Termination
   • Elongate: 72°C, 10 mins
   • Hold: 4°C, until removed from machine
   For long amplicons, elongation time = 1 minute + 2.5 s per 100bp.
   For shorter amplicons, under ~1kb, this can be shortened judiciously.
   

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 28 mins