Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )
Thank you for your replies. It turns out the carry-over contamination may infact have been the problem. After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem) the streaking has disappeared and the bands are bright.
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab. Very helpful.
Have you tried decreasing the Taq amount? Sometimes it can cause smearing too .
I'm aplying cDNA-RDA tecnique in bacteria and I am having the same problem. After the cDNA synthesis, it is digested and it is ligated to 24-mer adaptors. The cDNA digested and ligated to the 24-mer adaptor is amplified by pcr. Generally, this PCR uses 4mM MgCl2, a lot of Taq and the annealing temperature is 72oC. Analysing the pcr product by electrophoresis, there is a generalized smear extending whole lane. A 1kb-200pb smear is expected, but not the way I found. I tried reducing MgCl2 concentration to 2,5-3mM, number of cycles to 17 and the template concentration, but the problem persists. I don't know what to do. Somebody help me, please!
Make classical 3 temperature PCR and SHORTEN all HOLDS
How important is it to change the TAE buffer used to run the gels? and how often should I change it?
I have had this problem before in the lab. But we realised that it was because People in the lab were too lazy to change the TAE buffer that was used to run the gel.
Hope this helps!
I was wondering if anyone has answers as to why my PCR product is one big streak from well to end. Somtimes a band shows up where expected but in the background (or foreground) is the large, sometimes bright streak. We've been having this problem for a while. Sometimes it's not there but nothing different was done with extraction or PCR parameters. We use optimal thermocycling conditions for our primers, so we're pretty sure it's not in the PCR reaction.
We've also encountered bright bands in wells followed by those streaks of some samples. Any suggestions would be greatly appreciated!
Thank you in advance.
Do the size markers run ok. If they do the problem is in the PCR reaction. If the size markers look smeary then the problem is in the running buffer or gel.
If the gel.
I would replace the running buffer in the gel tank. Use the same freshly prepared buffer for the agarose gel too. In my experience the running buffer will last months before it needs to be replaced.
Maybe someone else in the lab switched to a different running buffer. TBE instead of TAE for example.
I had also same problem before. I guess one of your primers is bad. So why don't you try nested PCR and the PCR with all primer combination. So that you may find which primer is bad. If it is okk, you may change the polymerase. In some cases pfu polymerase can not amplify but taq polymerase can amplify the same band..
If you are purifying the band for cloning purpose it is strongly recommended that you have to use new buffer but if you are just checking the PCR band there is no reason to be worried about this. You may use the buffer until it looks dirty.
I've got a smear now and I wonder if total RNA transcribed by random hexamers and primers spanning 500 bp is a good combination. Maybe the cDNAs are much shorter than that.