Team:University College London/Protocols/Week12/3

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IrrE PCR

Gene to be amplified Primers
irrE Forward primer:
STF1: 5'-atggggccaaaagctaaagctgaagcc-3'
Reverse primer:
ST2R: 5'-tcactgtgcagcgtcctgcg-3'
irrE Forward primer:
STF3 5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3'
Reverse primer:
ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'

All primers were prepared to a concentration of 1pmol.uL, from 100pmol.uL stocks. Primers ordered from MWG operon.

Component 20 µl reaction 50 µl reaction Final concentration
Nuclease-free water to 20 µl to 50 µl
5X Phusion HF buffer 4 µl 10 µl 1X
10 mM dNTPs 0.4 µl 1 µl 200 µM
10 µM forward primer 1 µl 2.5 µl 0.5 µM
10 µM reverse primer 1 µl 2.5 µl 0.5 µM
Template DNA variable variable < 250 ng
DMSO (optional) (0.6 µl) (1.5 µl) 3%
Phusion DNA polymerase 0.2 µl 0.5 µl 1.0 units/50 µl PCR