Lab 11: Gel Analysis of PCR 2 (COX1 and V4)
Purpose: To run our controls and experimental DNA through the gel (gel electrophoresis) and analyze the success of COX1 and V4 primers using PowerSoil and Chelex.
Procedure:
- Remove rubber stoppers from gel mold and carefully put gel in gel electrophoresis chamber. Be sure the wells are closest to the black (negative) side of the chamber as DNA will run towards the positive end.
- Using the pre-made 10X TAE buffer solution, cover and fill the gel chamber until gel is fully covered.
- Add 5 ul of loading buffer to the positive/negative control samples as well as the experimental sample for both V4 and COX1.
- In the first well, carefully micropipette 5 ul of ladder. Add 10 ul of each PCR sample into individual wells (there should be 7 wells total).
- Put the lid on top of the gel chamber. Plug in the positive wire to the red side of the chamber and the negative to the black side of the chamber.
- Turn on the electrophoresis machine and set it at 95 volts for 30 minutes. Turn off the machine and carefully remove the gel wearing gloves.
- Slide gel off holder into UV light machine in order to take a picture of the DNA in the samples. We used a BioRad Gel Doc EZ System. Compare the results of the V4 and COX1 primers in the experimental DNA sample.
Results
There ended up being a good amount of visible DNA under the UV light for the COX1 primers for the groups who used the PowerSoil protocol. The Chelex protocol ended up giving good V4 product to be sequenced. For my group, we followed the PowerSoil MOBIO protocol and our results contained DNA, however there were signs of nonspecific binding. The bar where the V4 primer should have been was not visible indicating the primer did not bind very well with our DNA. However, the bar where the COX1 primer should have been was visible, but was located further away from the expected position. The COX1 primer binds to a larger region of the DNA and therefore, the bar should have been located closer towards the well because the DNA would be heavier than what the V4 primer would have binded to.
