I ran a PCR. My primers were specific to the region I wanted to amplify. I see the specific band (size 800 bp) and also a second band (size approx 1500 bp) in the agarose gel. Since I was curious about the identity of this second band, I gel extracted both bands and sent them for sequencing. To my surprise, both bands have the exact same sequence! Why are they running at two different levels in the gel?
it may be that the 5' end of one strand of the 800 base product and the 3' end of the other strand have some homology ( say 50 bases in from the ends) so the 800 product melts then re anneals with a 100 bp overlap. The taq will extend and make the whole product double stranded in the same way it does when extending primer annealed to the 800 product.
This will produce the sequencing effect that you see....one sequence perfect followed by about 700 bases of the same thing joined to it
Thank you all for your answers :) Yes these are all possibilities!
@ Michael, yes the primers I used for the PCR were the same that were used for Sanger sequencing.
maybe your forward primer is also complement to a secondary location upstream from the end of your target amplicon so your forward primers end up amplifying a region without need of the reverse primer. I suggest check the target sequence for possible matches. If it seems plausible you can test this hypothesis easily by using only one type of primer at a time.
that is a point Osman...it happens with primers with homology to repeat sequences which are very common and often occur in groups and sometimes the sequences face towards each other so one primer can amplify good bands. Here ,however, the template has been sequenced so I am assuming that the reverse primer also generates a sequence on both short and long products. Possibly Ambalika can confirm that sequencing was from both ends
What is the source of your template (i.e. prokaryotic, yeast, metazoan)? Sounds to me like you're amplifying a heterozygous locus.
Or using low-stringency conditions. The Tm should only be 5ºC below the lowest Tm of the pair of primers used. You can also increase the salt concentration (which will also require an increase in the Tm—you can recalculate the Tm of primers using IDT's OligoAnalyzer—just change the salt concentration). Also, your extension time may be too long. Depending on the polymerase you're using, 1'/kb is standard (e.g. for NEB Taq). So I'd only extend for 48 seconds to yield your desired 800-bp product.
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