PrbbBB:colony pcr v1

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Contents

Overview

Materials

Procedure

II PCR reaction

100µl single reaction 3.5xMaster 10xMaster
H2O 76µl 266 760
5x HF Buffer 20µl 70 200
10mM dNTP 2µl 7 20
FW primer 100 µM 0.5µl 1.75 5
RV primer 100 µM 0.5µl 1.75 5
Phusion 1µl 3.5 10
template DNA 0.1µl -- --
PCR Program
30"@98°C
5x (10"@98°C; 15"@Ta; text@72°C);
25x (10"@98°C; text@72°C);
10'@72°C
∞ 4°C
  • extension time text = (kb insert length) × 25"
  • annealing temperature Ta = (primer annealing) + 3°C

II Agarose Gel

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: no comment


References

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