| Troubleshooting with the Hive Mind [evolgen] |
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Written by 2000l, May 31st, 2007
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Late last week, my PCRs stopped working. One day I was able to amplify DNA from multiple different templates using different primers, and the next day I couldn’t. This is a major setback for me — a huge chunk of the remaining work I need to complete for my PhD involves doing PCR. If I can’t get my PCRs to work, I’m royally screwed.

As soon as I couldn’t get any PCR products, I went into troubleshooting mode. I had just made a new batch of dNTPs, so those were the first thing to get replaced. No dice. I thought there might be something wrong with my water, so I grabbed a new bottle and made another batch of dNTPs. Nothing. I replaced my buffer solution. No luck. I made a new dilution of each primer. Nope. I bought new Taq DNA polymerase and new stock solutions of each dNTP, and I remade my dNTP solution again. Nada. I prepped a new dilution of my template DNA (and I had been testing my PCRs using multiple templates that had worked in the past), but that didn’t help. I tried multiple other primers, two different thermal cyclers (I’m currently trying a third machine tonight), different containers of tubes and tips, but none of it helps. When I run my gels, I still get no bands in any of my lanes.
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