Team:University College London/Protocols/Week12/3

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(Difference between revisions)
(Created page with "IrrE PCR {| class="bigtable" |- ! Gene to be amplified !! Primers |- | irrE || Forward primer: <br \> STF1: 5'-atggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: ST2R: 5'-tca...")
 
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! Gene to be amplified !! Primers
! Gene to be amplified !! Primers
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|-
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| irrE || Forward primer: <br \> STF1: 5'-atggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: ST2R: 5'-tcactgtgcagcgtcctgcg-3'
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| 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'
| irrE || Forward primer: <br \> STF3  5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3' <br \> Reverse primer: <br \> ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'
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| 10 mM dNTPs || 0.4 µl || 1 µl || 200 µM
| 10 mM dNTPs || 0.4 µl || 1 µl || 200 µM
|-
|-
-
| 10 µM forward
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| 10 µM forward primer || 1 µl || 2.5 µl || 0.5 µM
 +
|-
 +
| 10 µM reverse primer || 1 µl || 2.5 µl || 0.5 µM
 +
|-
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| Template DNA || variable || variable || < 250 ng
 +
|-
 +
| DMSO (optional) || (0.6 µl) || (1.5 µl) || 3%
 +
|-
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| Phusion DNA polymerase || 0.2 µl || 0.5 µl || 1.0 units/50 µl PCR
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|}

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