Troubleshooting Lab 5 PCR

Gel electrophoresis results of PCR product digested for different amounts of time with HaeIII.
Gel electrophoresis results of PCR product digested for different amounts of time with HaeIII.

By Ann-Marie Barry, ABE Ireland technical support specialist

Teachers who joined us for an ABE Ireland Lab 5 workshop in 2019 will recall that lab 5 wasn’t working properly. The method we took you through in the training was not producing the expected results, or any results at all. Obviously, that was very frustrating, and we were very sorry that teacher’s efforts weren’t paying off.

So, what did we do? As we are part of the ABE global network so are lucky to have the combined experience and expertise of other members within our reach. We discussed our problem with Patricia Schoeppner from ABE Germany, who is also a European implementation coordinator and master lab technician. She was kind enough to provide some feedback on our protocols, and also shared with us the protocols used by ABE Massachusetts who use the MiniPCR. Closer to home, we also sought advice from Catherine Moss, a core technologist at the Conway Institute in UCD and who looks after the PCR equipment there. 

After trying, testing, and combining aspects of these new methods, a visible PCR product was present on the gels! Progress!

However, ( ☹ ) as every scientist knows, there’s often more than one hurdle to cross when trying to experiments to work. The next hurdle was that this product was no longer visible on the gel after digestion with the restriction enzyme, HaeIII. In the methods shared with us, the digestion times used by others varied between five minutes and 60 minutes – a very big range. So, with our war paint on and ‘Eye of the Tiger’ playing in the background, I digested 11 PCR products with the restriction enzyme with 11 different times five minutes apart.

Current Covid19 restrictions in the lab meant I could not work with others, so I could only use my own DNA for this troubleshooting. I am a homozygous taster (TT) so the results below will only show variations of the two expected bands expected from a TT taster.

After running many, many gels I determined that the optimum digestion time with HaeIII was 25 minutes. The ABE Ireland Lab 5 teacher's manual has been rewritten with the new protocol and optimised digestion time. We are delighted to share our adjusted protocols with teachers who have already attended a PTC PCR workshop. I have emailed teachers a note about this. If you haven’t received that from me, please let me know. Now that we have a working method, we’ll organise a date soon to share this workshop with any teachers who attended the Lab 1-4 workshop and would like to dive deeper with Lab 5.

Determining PTC genotype from gel-electrophoresis results