Team:Washington/GENERAL PCR PROTOCOL

From 2013.igem.org

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<b>General Digestion Protocol in PCR Tubes</b>
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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>
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<p dir="ltr">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></p>
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2013 UW iGEM team uses Xba1 and Pst1 <a href = "https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1=%7B2DAF5F0E-59C4-4A5D-8809-6CEAD6BAE4B4%7D&enzyme2=%7B90EFE184-8442-45F0-8286-04CA22EEFFF2%7D/">combination</a>for most digestions
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<br />
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<br>
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<p dir="ltr">Resuspending primers:</p>
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XbaI and SpeI together causes 50% inverted insertions and XbaI is less expensive than SpeI.
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<p dir="ltr"><a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">protocol: resuspending PCR primers</a></p>
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<br>
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<br />
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<br>
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<p dir="ltr">Working Stock: </p>
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<b>Digestion:</b>
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<p dir="ltr">(serves as a backup)</p>
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<br>
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<p dir="ltr">Dilute to 10 times the number on primer vial</p>
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10-42.5 uL DNA (200ng/uL = 2ug) Use the highest concentration (>50ng/ul) DNA available.*
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<p dir="ltr">10ul fwd primer 90ul DI water</p>
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<br>
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<p dir="ltr">10ul rev primer 90ul DI water</p>
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DI water to 50 uL (33.5ul for single digest or 32.5 uL for double digest)
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<br />
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<br>
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<p dir="ltr">Notes for iGEM 2013 HSB lab:</p>
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5uL of 10X buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) Use CutSmart with all ‘HF’ enzymes and XbaI.<br>
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<p dir="ltr">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. </p>
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0.5 uL BSA (If required. Not required for CutSmart. See above link) **<br>
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<p dir="ltr">Use the protocols below for Green Taq polymerase. </p>
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1uL enzyme 1 (kept on ice or freezer block)<br>
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<p dir="ltr">Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).</p>
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For double digest add:<br>
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<p dir="ltr">If PCRing insert use 1ng/ul DNA concentration.</p>
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<u>1uL enzyme 2 (kept on ice)***</u><br>
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<br />
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Total 50ul<br>
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<p dir="ltr">Amplification PCR with Green Taq polymerase:</p>
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(Enzymes can added last so they go into a 1X buffer concentration)<br>
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<ol>
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<u>Mix the reaction *well*</u> by pipetting up and down a few times (to mix the 50% glycerol).<br>
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  <li dir="ltr">
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<u>Incubate at 37C</u> for the required amount of time, usually 1 hr. Digests overnight work well.<br>
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    <p dir="ltr">Setup Amplification PCR Reaction</p>
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<br>
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  </li>
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  <ol>
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If double digest is required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.
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    <li dir="ltr">
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<br><br>
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      <p dir="ltr">1uL Template DNA</p>
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    </li>
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*1 unit of enzyme is defined as the amount needed to fully digest 1 ug of DNA in a 50 ul reaction in 1 hour. You can calculate how much enzyme and time is needed to do ~10-fold overdigestion.<br>
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    <li dir="ltr">
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**CutSmart contains BSA and is otherwise equivalent to Buffer 4 (except for DTT).<br>
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      <p dir="ltr">22ul deionized (dI) H2O</p>
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***Note that the enzymes are stored in 50% glycerol. The total RXN needs to be no more than 5% glycerol to get effective digestion. Therefore, the volume of enzyme you add should be no more than 10% of the total reaction volume. <br>
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    </li>
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****Thawing of buffer goes quickly when partially immersed in water. <br>
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    <li dir="ltr">
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<br>
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      <p dir="ltr">1ul 10uM forward primer (Tm XX C)*</p>
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See also:<br>
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    </li>
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<a href = "http://bio.freelogy.org/wiki/Restriction_Digestion"</a>
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    <li dir="ltr">
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      <p dir="ltr">1ul 10uM reverse Primer (Tm YY C)*</p>
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<br><br><br>
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    </li>
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Diagnostic Digest: use 10-20 uL Total
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    <li dir="ltr">
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<br>
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      <p dir="ltr">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>)</p>
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    </li>
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<table border="1">
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    <li dir="ltr">
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<tr>
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      <p dir="ltr">Total 50ul</p>
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<td>15 ul</td>
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    </li>
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<td>25 ul</td>
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  </ol>
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</tr>
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  <li dir="ltr">
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<tr>
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    <p dir="ltr">Amplification PCR Reaction Program</p>
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<td>10-20 uL of DNA<br>
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  </li>
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2.5uL 10x Buffer<br>
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  <ol>
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(0.5uL of BSA)<br>
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    <ol>
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0.5 - 10 uL of Enz 1<br>
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      <ol>
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0.5 - 10 uL of Enz 2<br>
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        <li dir="ltr">
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Fill to 25 uL with H20 <br>
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          <p dir="ltr">95 C for 30s</p>
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25 uL Total</td>
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        </li>
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<td>11 uL of DNA<br>
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        <li dir="ltr">
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1.5 uL of buffer 10x<br>
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          <p dir="ltr">95 C for 30s</p>
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(0.5 uL of BSA)<br>
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        </li>
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0.5 - 1.0 uL of Enz 1<br>
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        <li dir="ltr">
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0.5 - 1.0 uL of Enz 2<br>
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          <p dir="ltr">ZZ* C for 30s (important step)</p>
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15 uL Total</td>
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        </li>
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</tr>
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        <li dir="ltr">
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</table>
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          <p dir="ltr">72 C for 1min per kb target gene plus 10-15% (important step) </p>
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        </li>
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        <li dir="ltr">
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</p>
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          <p dir="ltr">Repeat 2-4 34x</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">72 C for 5 to 10min</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">4 C for forever (don&rsquo;t forget to turn the machine off the next day)</p>
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        </li>
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      </ol>
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    </ol>
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  </ol>
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</ol>
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<ul>
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  <li dir="ltr">
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    <p dir="ltr">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&rsquo;t have to be exact (within 5 degrees or so).</p>
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  </li>
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</ul>
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<br />
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<p dir="ltr">Analytical PCR with Green Taq polymerase:</p>
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<ol start="3">
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  <li dir="ltr">
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    <p dir="ltr">Setup PCR Reaction</p>
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  </li>
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  <ol>
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    <li dir="ltr">
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      <p dir="ltr">1uL Template</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">2ul dI H2O</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">1ul 10uM forward primer (Tm XX C)*</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">1ul 10uM reverse Primer (Tm YY C)*</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">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>)</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">Total 10uls</p>
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    </li>
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  </ol>
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  <li dir="ltr">
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    <p dir="ltr">PCR Reaction Program</p>
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  </li>
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  <ol>
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    <ol>
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      <ol>
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        <li dir="ltr">
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          <p dir="ltr">95 C - 30s</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">95 C - 10s</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">ZZ* C - 10s (important step)</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">72 C - 1min per kb target gene plus 10-15% (important step) </p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">Repeat b-d 25x</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">72 C - 5 to 10min</p>
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        </li>
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        <li dir="ltr">
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          <p dir="ltr">10 or 4 C - forever</p>
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        </li>
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      </ol>
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    </ol>
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  </ol>
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</ol>
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<br />
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<p dir="ltr">Phusion polymerase:</p>
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<ol start="5">
 +
  <li dir="ltr">
 +
    <p dir="ltr">Setup Amplification PCR Reaction</p>
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  </li>
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  <ol>
 +
    <li dir="ltr">
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      <p dir="ltr">1uL Template</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">1uL 25mM dNTP's</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">10uL Phusion HF Buffer</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">1.0ul Forward Primer (Tm XX oC)*</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">1.0ul Reverse Primer (Tm YY oC)*</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">0.5uL Phusion polymerase</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">36.5uL diH2O</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">Total 50uls</p>
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    </li>
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  </ol>
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  <li dir="ltr">
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    <p dir="ltr">Amplification PCR Reaction Program</p>
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  </li>
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  <ol>
 +
    <li dir="ltr">
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      <p dir="ltr">98 C - 30s</p>
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    </li>
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    <li dir="ltr">
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      <p dir="ltr">98 C - 10s</p>
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    </li>
 +
    <li dir="ltr">
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      <p dir="ltr">ZZ* C - 10s (important step)</p>
 +
    </li>
 +
    <li dir="ltr">
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      <p dir="ltr">72 C - 30s per kb target gene plus 10-15% (important step) </p>
 +
    </li>
 +
    <li dir="ltr">
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      <p dir="ltr">Repeat b-d 29-34x</p>
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    </li>
 +
    <li dir="ltr">
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      <p dir="ltr">72 C - 5 to 10min</p>
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    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">10 or 4 C - forever</p>
 +
    </li>
 +
  </ol>
 +
</ol>
 +
<ul>
 +
  <li dir="ltr">
 +
    <p dir="ltr">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&rsquo;t have to be exact (within 5 degrees or so).</p>
 +
  </li>
 +
</ul>
 +
<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>
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Revision as of 03:01, 21 September 2013


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<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Untitled Document

General PCR Protocol

See also: PCR_GoTaq and GoTaq(R) DNA Polymerase Product Information Sheet ... - Promega


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:

http://www.neb.com/nebecomm/products/protocol87.asp


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