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Team:British Columbia/Notebook/Protocols/PCR
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1. Make master mix of PCR components except the DNA. Keep on ice. | 1. Make master mix of PCR components except the DNA. Keep on ice. | ||
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1x (µL) x N ( µL) | 1x (µL) x N ( µL) | ||
Revision as of 23:58, 27 September 2013
>
iGEM Home
Colony PCR
Supplies needed:
PCR tubes
BioBrick PCr primers (VF2, VR)
Taq polymerase
10x Reaction Buffer
10mM dNTPs
dH2O
Colonies
Steps:
1. Make master mix of PCR components except the DNA. Keep on ice.
1x (µL) x N ( µL)
dsH2O 18.35 x N 10x rxn buffer 2.5 x N 10mM dNTP 1 x N 10mM FW primer VF2 1 x N 10mM RE primer VR 1 x N DMSO 1 x N DNA - - Taq 0.15 Total 25 x N
N=number of samples
- Add about extra amount worth two samples to account for water control and pipetting error.
(if regular PCR is done use ca.50-200ng template DNA and use Phusion and 10x Phusion buffer instead of Taq) 2. Aliquot 25µL per PCR tube, keep tubes on ice.
- Following steps must be done near the flame*
3. Touch toothpick/ pipet tip/ loop tp colony, then index plates, then dip in the PCR tube.
- Turn of flame*
4. Run PCR:
- Turn machine on
- Load samples in machine
- Select appropriate temperatures and times
1. 95°C for 10s
2. 94°C for 30s
3. 56°C for 30s
4. 68°C for 1min per kb of expected product (round up)
5. Repeat 2-4 30times
6. 68°C for 20 mins
7. 4°C forever
5. Once finished, remove the PCR tubes from the machine.
6. Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.
