Multiple Bands after gel extraction - (Nov/12/2013 )
Wondering if anyone can help me.
I'm trying to preare some samples for 454 sequencing. The DNA template is made up of various organisms but PCR primers are designed to amplify a only a 500bp of the 18s rDNA from all these different organisms (it does I've checked on individual organisms). So I get a strong 500bp band (50ng/ul and load 25ul), cut it gel extract and then run a 1ul sample to check quantity and I get two bands! One 500 and one 350bp. I've tried reducing the amount of PCR product I load before I get extract and PCR purification before I run the gel but I still get the same problem.
Hope you can help,
Just to add I've run it on a 2% and 1% gel for the first gel before gel extraction and the 350bp band is not visible when I cut the bands. There is slight smearing around the band as well but the 500bp band is pretty well defined.
Thanks in advance
May I know how the smear of your first gel look like? It is possible that 350bp band exist in a very light condition which could not be seen without purification. This happened might due to the presence of other gene which shows almost similar sequence as your target gene. Thus, primer will bind with it as well.
though I didn't get your second post, can it be unspecific amplification due to not sufficiently stringent conditions? I.e. I'd try out hot start, higher annealing temperature or less Mg2+ to reduce or disappear the unwanted band.
Just an update to my problem. It turned out that the second band only appeared after the ethanol precipitation. So I replaced this step with a PCR purification (Qiagen) and the band didn't reappear. We're still not sure why the precipitation step caused this extra band but some people have suggested that over-drying the pellet can cause DNA degradation. And as the extra band was half the size of the desired band it may be that the precipitation was creating single-stranded amplicons. Personally I'm not sure if this is true because the pellet was still visible when I eluted so I don't think it was overdry but anyway my problem is solved.
Many thanks for all your help guys,