Friday, July 4, 2008

Finally a proper PCR result in Gel Electrophoresis

I finally got what I wanted out of my PCR (polymerase chain reaction) and gel electrophoresis. I managed to find out the source of contamination that affected my results.

This was the first gel I did when I used my primers to amply a certain part of the DNA that I extracted from my tachyzoites. The first lane is the marker with ladders that will tell you the size of the product that we got. According to the gel, I have the right product because the size of my product is 720 bp(basepairs), however the control also has the same band in the same lane. Control has no DNA sample in it so it supposed to be clear. This gel could not be used for verification because we don't know if we got the real product or if it is the source of contamination.
I decided to troubleshoot the system to find out which component of my PCR was contaminated. I took the easy route by using a PCR mastermix, primers (foward and reverse) and deionized distilled water (DDH2O). So only these 4 components I need to check whether I accidentally contaminated them with my samples. I thought it might be the water so I used old DDH20, new DDH20 and sterile water. The result I got was so bad, all three lane were contaminated. I have no idea what to do, so I started to ask around on which area should I look upon to detect the source of contamination. My friends said to check each component of my PCR mix (mastermix, primers and water), autoclave the tips, autoclave bullet tubes, and have dedicated pipettes. I also planned to change gloves often, turn on the laminar air flow, stop scratching my self, and pretend I am the last person on earth so I won't talk to anyone.

TADA!!!!! no bands, which mean there is no contamination with my master mix, water or primers. So I am guessing either dirty tips, contaminated PCR tubes or just plain carelessness. Well I need to run a new PCR and new gel so that I can get a good picture that I can put in my thesis..
Post a Comment