Team:Washington/GENERAL PCR PROTOCOL

From 2013.igem.org

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<html>
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{{:Team:Washington/WA_Template}}
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<style type="text/css">
 
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.firstHeading {                                                    /* Gets rid of the default iGEM picture*/
 
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#menubar {
 
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#menubar ul li a {
 
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    color: #545353; }                                            /* menu text color*/
 
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#siteSub {
 
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#contentSub {
 
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h1
 
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h2
 
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h3
 
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p
 
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<strong id="docs-internal-guid-13513859-3e76-8503-3f74-96146348d33f">
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<h1 dir="ltr">General PCR Protocol</h1>
<h1 dir="ltr">General PCR Protocol</h1>
-
<p dir="ltr">See also: <a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">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></p>
+
<p dir="ltr">See also: <a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">PCR_GoTaq</a>
<br />
<br />
<p dir="ltr">Resuspending primers:</p>
<p dir="ltr">Resuspending primers:</p>
-
<p dir="ltr"><a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">protocol: resuspending PCR primers</a></p>
+
<p dir="ltr"><a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">Protocol: Resuspending PCR primers</a></p>
<br />
<br />
<p dir="ltr">Working Stock: </p>
<p dir="ltr">Working Stock: </p>
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<p dir="ltr">Refer for further details to NEB's online protocol for Phusion:</p>
<p dir="ltr">Refer for further details to NEB's online protocol for Phusion:</p>
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<p dir="ltr"><a href="http://www.neb.com/nebecomm/products/protocol87.asp">http://www.neb.com/nebecomm/products/protocol87.asp</a></p>
+
<p dir="ltr"><a href="http://www.neb.com/nebecomm/products/protocol87.asp">Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase</a</p>
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Latest revision as of 04:31, 27 September 2013


Untitled Document

General PCR Protocol

See also: PCR_GoTaq

Resuspending primers:

Protocol: Resuspending PCR primers


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 (GoTaq) 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:

  1. Setup Amplification PCR Reaction

    1. 1uL Template DNA

    2. 22ul deionized (dI) H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 25ul 2x Green taq (GoTaq)

    6. Total 50ul

  2. Amplification PCR Reaction Program

        1. 95 C for 30s

        2. 95 C for 30s

        3. ZZ* C for 30s (important step)

        4. 72 C for 1min per kb target gene plus 10-15% (important step)

        5. Repeat 2-4 34x

        6. 72 C for 5 to 10min

        7. 4 C for forever (don’t forget to turn the machine off the next day)

  • 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:

  1. Setup PCR Reaction

    1. 1uL Template

    2. 2ul dI H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 5ul 2x Green taq (GoTaq)

    6. Total 10uls

  2. PCR Reaction Program

        1. 95 C - 30s

        2. 95 C - 10s

        3. ZZ* C - 10s (important step)

        4. 72 C - 1min per kb target gene plus 10-15% (important step)

        5. Repeat b-d 25x

        6. 72 C - 5 to 10min

        7. 10 or 4 C - forever


Phusion polymerase:

  1. Setup Amplification PCR Reaction

    1. 1uL Template

    2. 1uL 25mM dNTP's

    3. 10uL Phusion HF Buffer

    4. 1.0ul Forward Primer (Tm XX oC)*

    5. 1.0ul Reverse Primer (Tm YY oC)*

    6. 0.5uL Phusion polymerase

    7. 36.5uL diH2O

    8. Total 50uls

  2. Amplification PCR Reaction Program

    1. 98 C - 30s

    2. 98 C - 10s

    3. ZZ* C - 10s (important step)

    4. 72 C - 30s per kb target gene plus 10-15% (important step)

    5. Repeat b-d 29-34x

    6. 72 C - 5 to 10min

    7. 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:

Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase

Retrieved from "http://2013.igem.org/Team:Washington/GENERAL_PCR_PROTOCOL"