pALS PCR 3 results

Blah! I grow disdain for failed PCR reactions. I have no idea what the issue is and I hate troubleshooting PCR. Oh well. I’ll stop venting and get right back to work.

So the PCR was a failure again. And now I have to figure out how to adjust it. Since I’m not getting any product here are my potential fixes:

  • adjust the annealing temperature
  • try different amounts of MgCl2
  • try new dNTPs

In order to properly adjust the annealing temp I would need to make sure the machines are functioning properly, which I’m figuring they aren’t. The reason I think this is because when I check the thermal cycler (not OpenPCR) it says Program end in 0:00 and usually says Hold 4C. But the reason I think the thermal cyclers are not the issue is because neither of them produce product, their reactions are identical, and their temps are very similar.

In order to troubleshoot the machines I’ll have to run a temp experiment where I track the temperatures over time. I have equipment for this, but I can’t find all the components. I’ll have to talk to Pranav about the missing parts.

Trying different amounts of MgCl2 is easy. I would just do amounts ranging from 1 to 5mM of MgCl2 all on the same program.

In order to try new dNTPs I would just need to buy new dNTPs. This can’t happen until Monday. Neither can the thermal cycler experiment. Tomorrow I can try a MgCl2 titration experiment. Right now I’ve got a simple check in mind.