Team:Groningen/protocols/Colony PCR

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Colony PCR

Materials:
  • PCR tubes
  • MQ water
  • DMSO 5%
  • Phusion buffer
  • DNTPs
  • F- and R-primer
  • Phusion Polymerase
  • DNA template


PCR reaction mixture:
Component 20 µl Final concentration
MQ water up to 20 µl
5x Phusion Buffer 2 µl 1x
DMSO 5% 1.5 µl
10 mM dNTPs 1 µl 200 µM
primer F 1 µl 0.5 µM
primer R 1 µl 0.5 µM
template DNA 1 µl ∼1 ng
Phusion Polymerase
2U/µl		 0.3 µl 0.01U/µl


Cycling instructions:
Temperature Time Number of Cycles
98°C 10 min 1
98°C 10 sec 34
Tm-5°C 25 sec
72°C 30 sec/kb
72°C 10 min 1
4°C ∞ 1