When amplifying DNA from a plasmid by PCR (in order to add some restriction sites for cloning), we see a shift from the expected target band when using 1 µg template DNA, to increasingly higher amounts of an unspecific band at 100, 10 and 1 ng template DNA. All other parameters of the PCR were kept constant. Since I never saw this before during PCR, can anyone explain why fewer template DNA can lead to higher amounts of a defined unspecific band? Thank you in advance.
Hi, Benjamin. I think it's most likely primer dimers or other weird products caused by primers mis-annealing. When there's not enough template, the primers have nothing to do, and can start to cause mischief. Try decreasing primer concentration, or reduce the number of cycles, or increase the annealing temperature. Those might help reduce unwanted primer interactions.
Hi, Benjamin. I think it's most likely primer dimers or other weird products caused by primers mis-annealing. When there's not enough template, the primers have nothing to do, and can start to cause mischief. Try decreasing primer concentration, or reduce the number of cycles, or increase the annealing temperature. Those might help reduce unwanted primer interactions.
Ivan is correct in that sequences within primers will always bind to non target material and potentially amplify in the absence of a "correct" target ( including other primers).
1ug of plasmid DNA seems a lot of material to start with in an amplification. For a 5kb plasmid it is equivalent to nearly 2e11 copies and will very quickly reach saturation - how many cycles are you doing? At saturating amts of target incomplete linear products of increasingly smaller sizes will be generated with increasing cycles.
As you have added RE sites to your primers it will affect the Tm of the primer - the Tm calculated on the data sheet is based upon a 100% target sequence identity whereas your primer may for example be 35 bp long including a non-identical sequence of 10 bp (6 RE sequences and a 4nt leader). Paradoxically you may have to lower your annealing temp at least for the first few cycles and ramp up to get good amplification (Tm based upon the actual sequences that anneal to the target). At very high concn of target, a poor binding efficiency will still generate product. As the target number decreases the correct amplification will also decrease but can still reach saturation. If you do these reactions in a real-time PCR machine you can observe the rise of amplicons and reduce the number of cycles to generate sufficient product without some of the artefacts.
Ian
Although primer dimer is a probable explanation, depending on size, another is a contaminant that is being introduced with one of the reactants other than template. As the template is reduced the contaminant out competes it. This will give a second band.
But even for complex templates such as whole mammalian genomes 1ug of template is more than is optimum for most PCR. The number of somewhat mismatched sites for which the primers have some affinity sites is so large that a lot of primer can be mopped up, and gives a light background smear. Primer dimer is reduced, but so is the correct product.
For a plasmid I would certainly drop the concentration at least 1000 fold as a starting point.
as said above try to decrease primer concentration with decrease in Template concentration.....
All the best
DNA binds Mg ions, so the free Mg concentration rises when adding less template DNA. Higher Mg concentration gives more aspecific primer binding and as a consequence more aspecific PCR products are formed.
1000 ng of starting amount of the template DNA is very high, 10-20 ng is a better range to use. High amount of starting material gives saturation of the PCR within a few cycles, when a concentration of about 10.000 ng is reached, the polymerase is inhibited and stops working. When lower amount of starting material is used the polymerase is active in more PCR cycles and the chance of aspecific PCR products increases every cycle.
The chance of primer dimer formation also increases with Mg concentration and cycle number. Primers with restriction sites are 10 bp longer then normal, the chance of complementary sequences between the primers increases by lenght, which enhances the chance of primer dimer formation.
Using hotstart prevents primer dimer formation to a large extend. So add the polymerase when the tubes are in the thermocycler at the annealing temperature, 60 degrees Centrigrade or higher, depending of the melting temperature of your primers.
Thank you all for your answers!
Just to clarify: I am aware that 1 µg starting material is of course not a good choice and we actually only compared different amounts of 1-1000 ng as an exercise for one of our students. Nevertheless, I thought the unexpected outcome was kind of interesting and I am happy to having received your helpful comments!
I agree with Benjamin that 1 µg starting material too much for PCR. I think you should also try different annealing temperature..
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