Team:University College London/Week12YanikaExp4


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In order to prepare the Deinococcus radiodurans template for PCR, a colony was picked off a plate (supplied by Prof. John Ward), resuspended in 10uL dH2O and added to boiling water in a screw top vial. This was left to return to ambient room temperature.

The primers used are listed in the table below. All primers were ordered from MWG operon and were prepared to a working concentration of 1pmol/uL from 100pmol/uL stocks.

Primer Sequence
Forward primer - STF1 5'-atggggccaaaagctaaagctgaagcc-3'
Reverse primer - ST2R 5'-tcactgtgcagcgtcctgcg-3'
Forward primer(with Biobrick prefix/Suffix) - STF3 5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3'
Reverse primer(with Biobrick prefix/Suffix) - ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'

PCR was preformed using NEB High Fidelity polymerase. The following 20ul and 50ul reactions were set up as follows:

Component 20 µl Reaction 50 µl Reaction Final Concentration
Nuclease-free water to 20 to 50 ul -
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 1ul 1ul < 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