So, I am trying to amplify a 1000 bp sequence from genomic DNA. I've amplified 5 other regions of the same genomic DNA, also on 1000 bp, which have worked perfectly.
What happens is:
I make a master mix of water, polymerase buffer, dNTP, and polymerase (adding the pol as last reagent) before adding to the tubes (which already contain primers and genomic DNA). I normally have 6 tubes with different primers for different regions. 5 of them works every time. The 6th doesn't work no matter what I do.
What I have tried so far:
Polymerase: using either taq, phusion or a combination.
Primers: making new primers a few basepairs up or down from where I made the first primers. Making sure there are no long repeating sequences. Making different length (but at least 15 bp long).
Genomic DNA: making fresh batches of genomic DNA, diluting the DNA, having a high concentration of DNA.
PCR Cycler: using annealing gradient of 45-60 C, using different elongation time, adding primers after a few circles, adding tubes after heating the cycler.
I am not sure what else I can do to try and get something out of this sequence. I need it to silence the gene which comes right after the sequence.
On the picture here you can see all the sequences tested for optimal annealing temperature. The sequence which didn't work is the first 4 lanes. I have other gels where this specific sequence only shows as a smear on the bottom of the gel, or not at all.
Hope you guys have some other ideas of how to proceed. Thanks! :)
My first impression is that you have a large intron. Try using a polymerase and kit for amplification of 10 kb stretches. Obviously your DNA is good quality, since the other regions amplify.
Hi Sanne,
have you tested DNA from different samples? Are you sure that the target is not altered in the analyzed sample(s)? To get some more information you may sequence the "unspecific" PCR products. Furthermore, if you know the complete sequence you can try to amplify in several smaller PCR products.
Good luck
Hi Sanne,
Norman gave you good suggestions. It is important that you use more than one sample as template. Have you checked for the presence of SNPs in primer sequences?
If it is not fundamental that you use exactly those primers, I would try to design new primers near those that are not working, moving up or down.
good luck!
Is it a GC rich area? Usually adding DMSO or betaine help with complicated PCR from genomic DNA. The denaturation times should also be appropriate. When phusion doesn't work I've had sucess with LongAmp from NEB (a mix of TAQ and Q5). If nothing works definetelly new primers or consider synthesis (its getting cheaper by the day)
Hello Sanne,
I guess you already got a lot of good suggestions and perhaps by now you've figured it all out. So I will just briefly share what was my first impression when I saw the gel picture.
It does look like you have multiple bands, so you are getting more fragments amplified than you hoped for. As you know from the other samples, the DNA itself is not fragmented, so would it be possible that you have multiple primer binding sites, perhaps some where the primer does not match perfectly?
Have you also tried a hot start PCR to ensure that the polymerase only works when it is supposed to and not already a little bit all the time?
PCR can be super-frustrating, but I hope you will figure it out!
best, ac
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