Module 2, Lab 05 - GAPDH Nested PCR

Today we ran a gel electrophoresis to confirm the results of the second round of PCR (using the nested primers).

Ideally, we would have purified the Arabidopsis PCR product (GAPC gene; in fact, section 1 did follow the process), but all PCRs failed. WHY?

Here's what I think happened:

When I retrieved the tubes from the thermocycler they had a very small volume of liquid at the bottom of the tube. Most of the volume (pretty much all the water, not just the water you added) was condensed at the top of the tube. That means that all the reagents were desiccated and molecules couldn't interact with each other. Result: no reaction whatsoever.

Probable cause: A glitch in the thermocycler. Most thermocyclers today have heated lids, to prevent condensation of water at the top of the tube. There is evaporation, but by preventing condensation water is always being recirculated in between its liquid and gas states and there is always enough liquid water to keep the PCR going. If the lid doesn't heat up during the process, then most of the water evaporates, condensates at the top of the tube and the reaction is ruined.

I have no idea of why the lid wouldn't heat up, since it is an automatic process every time you run a program.

Solution: I am running the nested PCRs again. The first round is in the thermocycler as I type and I triple-checked to make sure the lid was hot. I will run the second round and a gel to make sure that we have product (GAPC gene). If the reaction works, in week 5, before the ligation and transformation exercise, you will have to purify the PCR product before proceeding.

Lesson: Learn how to deal with frustration. In molecular biology many things can go wrong when following a protocol and you must keep on going. If you ever become part of a research lab you will find out, first hand, that nothing is as perfect as it looks in the published literature. Today you had a little taste of it.

We must shake it off and do it again. In this case I have to do it again (but if there are any volunteers for setting up the second round of PCRs, let me know)

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Results of make up PCRs

The following gels show the results of the nested PCR (click on the pic to see a full size image):

Lanes 1 and 2 on both gels are the positive and negative controls, of the first round PCR on the left gel and of the nested PCR (2nd round) on the right. The box on the left gel shows a faint band, which resulted from leaking when I was loading the positive control in the adjacent well.

Lanes 3 and 4 on the left gel show products of the first round of PCR, and all other lanes in both gels show products of the second round of PCR.

I have saved the products of the nested PCRs for you to purify this week and go on with lab 6.

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