Team:Washington/GENERAL PCR PROTOCOL
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Revision as of 03:01, 21 September 2013
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General PCR Protocol
See also: <a href="https://docs.google.com/document/d/16Piw2IQOHUwHPN0zY0PJp5cm4c9gFu6VLiOj-1kspP8/edit?usp=drive_web">PCR_GoTaq</a> and <a href="https://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq(R) DNA Polymerase Product Information Sheet ... - Promega</a>
Resuspending primers:
<a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">protocol: resuspending PCR primers</a>
Working Stock:
(serves as a backup)
Dilute to 10 times the number on primer vial
10ul fwd primer 90ul DI water
10ul rev primer 90ul DI water
Notes for iGEM 2013 HSB lab:
We are using the GreenTaq polymerase 2X pre-mixed (<a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>) for PCR.
Use the protocols below for Green Taq polymerase.
Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).
If PCRing insert use 1ng/ul DNA concentration.
Amplification PCR with Green Taq polymerase:
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Setup Amplification PCR Reaction
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1uL Template DNA
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22ul deionized (dI) H2O
-
1ul 10uM forward primer (Tm XX C)*
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1ul 10uM reverse Primer (Tm YY C)*
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25ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)
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Total 50ul
-
Amplification PCR Reaction Program
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95 C for 30s
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95 C for 30s
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ZZ* C for 30s (important step)
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72 C for 1min per kb target gene plus 10-15% (important step)
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Repeat 2-4 34x
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72 C for 5 to 10min
-
4 C for forever (don’t forget to turn the machine off the next day)
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Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).
Analytical PCR with Green Taq polymerase:
-
Setup PCR Reaction
-
1uL Template
-
2ul dI H2O
-
1ul 10uM forward primer (Tm XX C)*
-
1ul 10uM reverse Primer (Tm YY C)*
-
5ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)
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Total 10uls
-
PCR Reaction Program
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95 C - 30s
-
95 C - 10s
-
ZZ* C - 10s (important step)
-
72 C - 1min per kb target gene plus 10-15% (important step)
-
Repeat b-d 25x
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72 C - 5 to 10min
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10 or 4 C - forever
Phusion polymerase:
-
Setup Amplification PCR Reaction
-
1uL Template
-
1uL 25mM dNTP's
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10uL Phusion HF Buffer
-
1.0ul Forward Primer (Tm XX oC)*
-
1.0ul Reverse Primer (Tm YY oC)*
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0.5uL Phusion polymerase
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36.5uL diH2O
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Total 50uls
-
Amplification PCR Reaction Program
-
98 C - 30s
-
98 C - 10s
-
ZZ* C - 10s (important step)
-
72 C - 30s per kb target gene plus 10-15% (important step)
-
Repeat b-d 29-34x
-
72 C - 5 to 10min
-
10 or 4 C - forever
-
Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).
Refer for further details to NEB's online protocol for Phusion:
<a href="http://www.neb.com/nebecomm/products/protocol87.asp">http://www.neb.com/nebecomm/products/protocol87.asp</a>
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