PCR contamination that comes and goes! - (May/23/2007 )
Hello,
I am having problem with contamination in my reaction. I am getting a band in my negative control in the PCR reaction that doesn't appear in the next PCR reaction using the same reagents, tips pippettes . I even tried a PCR reaction with 4 negative controls and the contamination appeared in the 2 controls and not in the other 2! Can someone give me a clue why this thing is happening?? I use genomic DNA and I am amplifying a human gene that is expressed in all samples. Please HELP!!!!
I am having problem with contamination in my reaction. I am getting a band in my negative control in the PCR reaction that doesn't appear in the next PCR reaction using the same reagents, tips pippettes . I even tried a PCR reaction with 4 negative controls and the contamination appeared in the 2 controls and not in the other 2! Can someone give me a clue why this thing is happening?? I use genomic DNA and I am amplifying a human gene that is expressed in all samples. Please HELP!!!!
Did you clean your PCR area well ? it might be contaminated from your glove or tube PCR. Is the sterility of your reagents, distilled water and pipett tips enough? If I were you, I would be prepared everything in the new set and then repeat the PCR reaction again. Avoid using the old thing. Try it again..hope you suscessful
krapom is right, try to clean up first.
PCR reaction is very sensitive. Contamination might just happen during preparation of master mix. Just be careful. 
If it appears in 2 out of 4 negative controls from the same master mix then it's probably not your reagents. I would blame your pipettes. Are you using filter tips?
I set up a reaction with filter-tips today to exclude pipette contamination. I am waiting for the results! Thank you all!
I remember I got the same problem before, and after cleaning the PCR area (Hood) very well and after sterilizing every thing using an autoclave (tips, PCR tubes and pipettes) I got the results 