Team:University College London/Protocols/Week12/3

(Difference between revisions)

Latest revision as of 22:02, 26 September 2012

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

@@ Line 5: / Line 5: @@
 ! Gene to be amplified !! Primers
 |-
-| irrE || Forward primer: <br \> STF1: 5'-atggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: ST2R: 5'-tcactgtgcagcgtcctgcg-3'
+| irrE || Forward primer: <br \> STF1: 5'-atggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: <br \> ST2R: 5'-tcactgtgcagcgtcctgcg-3'
 |-
 | irrE || Forward primer: <br \> STF3  5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: <br \> ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'
@@ Line 22: / Line 22: @@
 | 10 mM dNTPs || 0.4 µl || 1 µl || 200 µM
 |-
-| 10 µM forward
+| 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
+|}