PCR contamination with human DNA first and now no bands!!!
Posted 17 June 2009 - 12:23 PM
Then the bands appeared better but there was a contamination with the water control which was confirmed as human DNA using a ladder since it was parallel to my product (246bp). I changed all my tips, pipettes and even the reagents excluding the primers. I took a new dilution from the stock of primers and used it with no luck.
Then suddenly for the last 3 attempts I made there were absolutely no bands.
We even changed the running buffer (TAE) but still no luck.
After the PCR I usually purify it and do a RFLP using Hha I enzyme but after the contamination bands appeared I stopped the RFLP and also found that the problem is not with the purification step either because I changed those reagents as well. Therefore the problem got to be some where in the PCR.
I even used a DNA sample that is used as a control from a friend but still no bands appeared.
I've been trying to get some results for a really long time now (Around 4-5 months now!)
Please help! Really confused!!!
Thank you in advance for anyone who helps me at least with a small suggestion!
Posted 18 June 2009 - 02:10 AM
Things can go bad, especially dNTPs are very likely candidates, exchange your reagents and try again.
If it doesn't work then re-check your primer sequences and make sure they are supposed to bind where you think they should.
If this also doesn't work check your thermocycler / use another cycler.
Did you do a successful, reproducible PCR yet (a different PCR I mean)? If not your set-up might be the problem and you should ask someone to show you how it is done properly.
Posted 18 June 2009 - 07:36 AM
Posted 23 June 2009 - 11:33 PM
Posted 26 June 2009 - 09:38 PM
I did the PCR again with a new set of reagents and it worked out well. I think the reagents I used would have been contaminated some how even though it was a new set from the lab technician or maybe I contaminated them!