Barrick Lab :: ProtocolsTroubleshooting

---+ “My PCR didn’t work…What do I do?”&#8203;
---+++A guide to PCR troubleshooting

---++ Check your primers
   * PCR primers should be between 18-25 bp, though slightly longer is ok.&#8203;
   * It is recommended that primers begin and end on a C/G.&#8203;
   * It is recommended that primers be designed so that the annealing temperature shown on benchling is roughly 57-60°C&#8203;
   * Since Phusion is the most commonly used polymerase in the Barrick lab, make sure your melting temperature calculator in benchling is set to Phusion&#8203;
   * This can be accomplished by clicking on the melting temperature icon at the bottom of the screen and selecting Phusion setting from the drop-down menu&#8203;

---++ Check your annealing temperature – there are several methods&#8203;
   * Plug the portions of your primers that physically anneal to your template into the NEB annealing temperature calculator.  Then use that temperature.&#8203;
   * If that fails, and you are not seeing your desired band or any band for that matter, decrease the annealing temperature to 54C and run the PCR again.&#8203;
   * When running your PCR with a low annealing temperature such as 54C, you may start to see off-target bands on the gel.  If this happens, gradually increase the annealing temperature (if you ran it at 54C before, try running it at 57C this time) and repeat the PCR&#8203;
   * If all else fails, redesign your primers&#8203;

---++ Check your extension time&#8203;
   * NEB recommends an extension time of 30 sec/kb – To be safe, set your extension time to 1 min/kb+1 minute&#8203;
   * While this may seem excessive, it works.  The polymerase will not die or denature from adding an extra minute to your extension time.&#8203;
   * PCR_elongtime.png: <br />
      <img src="%ATTACHURLPATH%/PCR_elongtime.png" alt="PCR_elongtime.png" width="650" height="400" />


---++ Check the number of PCR cycles you are running&#8203;
   * Typically, 30-35 cycles are sufficient&#8203;
   * For particularly stubborn amplicons, try running at 40 cycles&#8203;
   * Do not exceed 40 cycles&#8203;
     * Even if you only had 1 molecule of template, it should have been amplified to a strong band with this many cycles! If not, then your PCR is very inefficient due to some problem (annealing temp, extension time, mistake in design, template plasmid is not what you thought, etc.).&#8203;
     * “Over-PCR” can result in off-target products forming when the strands of your product dsDNA start annealing to each other in ways that result in further extension. This is often but not always seen as bands at larger than expected sizes starting to accumulate.&#8203;
   * PCRprod_temp.png: <br />
     <img src="%ATTACHURLPATH%/PCRprod_temp.png" alt="PCRprod_temp.png" width="750" height="450" />

---++ Check your template&#8203;
   * Did you sequence the plasmid / genome you are using as template? Mix-ups happen and discrepancies between design sequences and actual sequences may cause failure.&#8203;
   * Plasmid concentrations should be between 1pg to 10ng in a 50uL reaction
   * Genomic DNA template concentrations should be from 50ng to 250ng in a 50uL reaction
   * If your template is a PCR product, do not run another 35 cycles!&#8203;
   * Instead, run the reaction with 10-12 cycles&#8203;
   * Using too much template or running another PCR off of a previous PCR product will cause smearing on the gel&#8203;
   * PCR_toomuchtemp.png: <br />
     <img src="%ATTACHURLPATH%/PCR_toomuchtemp.png" alt="PCR_toomuchtemp.png" width="800" height="420" />

---++ Check your PCR buffer&#8203;
   * Typically, with Phusion, the HF 5X NEB buffer is sufficient.&#8203;
   * For stubborn amplicons, or amplicons with >60% GC content, it is recommended to use the Phusion GC buffer&#8203;
   * Always add DMSO &#8203;

---++ Special Notes for Short PCR products (<250 bp)&#8203;
   * Beware of staining “dead spots&#8203;
   * The DNA stains (e.g., SYBRsafe) has a positive charge, so it will migrate ”upwards” in your gel.&#8203;
   * If you have small PCR products and run a gel for long enough, they can end up in the part of the gel that most of the gel stain has moved out of.&#8203;
   * This will result in a “false-negative” where you see a faint band for your product and think the PCR has failed, when it actually hasn’t!&#8203;
   * Beware of low yields from column-purification kits.&#8203;
   * Short dsDNA fragments do not bind well to these columns, so you will tend to get lower yields after PCR cleanup / gel extraction&#8203;
   * If you don’t care about getting rid of the primers, ethanol precipitation is an alternative that can be used.&#8203;


-- %USERSIG{PraneshRao - 2025-03-20}%
-- %USERSIG{AnthonyVandieren - 2025-03-20}%

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Barrick Lab > ProtocolList > ProtocolsTroubleshooting
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PraneshRao
Topic revision: r1 - 2025-03-20 - 22:27:49 - Main.PraneshRao