Bisulfite treated DNA specific primer set critique? - (Aug/18/2005 )
One more question for you nick. In the protocol that you posted the final resuspension step is in 50 uL. Do you only use 1 microliter of that suspension for the nested PCR?
Starting with 2ug and assuming you lose about 90-94% of the DNA through degradation in the bisulfite treatment which leaves you about .1-.2ug suspended in 50 uL. Thats about 2-4 ng of bisulfite treated DNA per pcr unless I'm mistaken? That's sufficient?
indeed it is, that is one of the reasons you require two rounds of pcr using heni nested primer sets
the force is strong with this one!!
Well I did the bisulfite treatment using your protocol, which worked great I think, and ran my nested pcr on my bisulfite treated samples. I got my expected product (~410 bp) on at least a few of my samples after running a TD PCR, but I am also seeing some fainter bands @ about 100-200 bp. I'm not really sure what to make of them, are they likely to just be background? They seem to be in almost every sample that I have my expected product in.
I am going to try to run a magnesium titration to see if that will eliminate them but do you have any other ideas on what might be causing them? I used a mg concentration of 2mM.
I'm attaching a picture of my gel if you can spare a couple of minutes to take a look at it. Two 100bp ladders are included, and I ran both sets of controls (from first and second round of PCR) on the gel as well. The first and seventh samples I wasn't really expecting bands on because I might have lost my pellet in the precipitation step, but a few of my samples which didn't have my amplified target were isolated using the Promega Genomic SV purification Kit and should have worked. Have you had any experience with DNA isolated from that kit?
It is great to see you are making progress cwong!!!
as for the spirous bands, can I ask of you are amplifying a unique gene sequence? because of the nature of the bisulfite reaction I would say it could be possible that degenerate primer sites within the genome may occur. Increasing the Tm may also get rid of the smaller bands, they are indeed small but a littl too big to be just primer dimer, something I usually get.
A magnesium titration could solve your issue, good luck!!!
out of interest, which lanes are your first round of PCR??
You bring up a good point. Since my primers were fairly long I had thought that the chances of running into a degenerate primer binding sequence resulting from bisulfite treatment were fairly slim especially when running a nested PCR. I will look into it but yes, the primers were designed for a unique gene sequence AFAIK.
The gel photo that I have posted were all 2nd round PCR samples (less two controls from the first round.) Since seeing those extra bands on the gel I was planning on running a gel for the first ound pcr products(if there are any) but just haven't had a chance to do it. Hopefully the magnesium titration solves my problems, I ran the nested PCR today and am running the gel right now.
Well, looks like i finally solved the problem with the smaller sized bands. The magnesium titration didn't help, but lowering my annealing temperature did. Oddly enough, my calculated Tm for the primers were 66 and 67 respectively but after running a gradient of annealing temps it appears that 59-60 was optimal, 6-8 degrees below my Tms, and eliminated all other bands besides my product. Are the calculated Tm's from perl primer usually correct?
I'm planning on running some restriction digests to QC for complete conversion in my sequence before I move on, but everything seems to be looking good. Thanks for all the help Nick.
That's great news cwong,
perlprimer uses the most current formula for calculating Tm, however it is not always correct, and I have had to lower my Tm by at the most 10C from the calculated Tm also.
All the best,
Confused So I had thought that I had nailed down the optimal annealing for my primers, but now I am having second thoughts. I tried testing another one of my bisulfite treated samples using what I had determined would be optimal based on the gradient PCR (which was done on a different sample). The band was very faint, so I tried another gradient and found the optimal annealing temperature for a strong band to be 2 degrees below my original finding. I retested my first sample with the new annealing temp but the band was much fainter .
So it would appear that my optimal annealing tempfor my primers is different depending on my samples? How can that be? Since my primers incorporate a good number of conversion events, would my results be an indication that my primers are having some issue binding due to incomplete conversion in some of my samples?
could very well be what you are saying cwong,
in cases like these, it could very well be the salt concentration within your PCR.
So choose a Tm, your original or the second one, and then perform a Mg salt titration between 0mM and 4mM final concentration.
most PCR buffers have a 1.5mM Mg by default and Mg concentration will affect the way your primers bind to the template!!!