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.
I can pretty much rule out the possibility that I’m getting amplification, but my gels are screwy for two reasons. First of all, my ladder runs perfectly fine in all the tests. Secondly, I reran some PCR products from a couple of weeks ago (back when I was able to perform a successful PCR), and they looked a-ok.
So I’m turning to you guys. Now that I have replaced every reagent, every solution, and my template, what can I do? Is there anything I haven’t thought of (tinkering with Mg2+ concentration, perhaps?) that will fix this problem? Keep in mind, I went from really nice PCR products to none at all, and my primers are designed to the genome sequence of the species and strain I’m amplifying. What’s a helpless grad student to do?