"

From 2012.igem.org

---

Revision as of 21:49, 26 September 2012

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