Inconsistent MSPCR - Inconsistent MSPCR (Aug/09/2005 )
Hello everyone. I've been getting some inconsistent PCR results lately. I am performing methylated specific pcr using a touchdown protocol in a GeneAmp 9700. The problem is that my PCR as of late has become a roll the dice event in that sometimes it works and others it doesn't given the same conditions and concentrations of reagents. I've tried playing with my primers switching dNTP and DNA concentrations. Another phenomenon
I've noticed with my reaction is that it works better if I don't mix the reaction after adding my DNA to the mastermix. If anyone has any comments, suggestions I would really appreciate your help. Thank you very much
Have a few questions for you gungrave.
1. have you also been using the same starting template DNA or is this from different preps of DNA?
2. Can you use a Template/Primer combination that works everytime with all your PCRs?
Once we get these answers I am sure we would be able to give you some suggestions
Thanks for your reply Nick.
I have been for the most part using the same starting template. The thing is I treat my DNA using the EZ Meth kit and I use the template from there until I run out and I have to treat some more. I do get my DNA for treatment from the same prep.
With the template/primer combination, we have some primers that have been optimized for normal DNA, would these do? or do they have to be specific for treated DNA
Another question if I may? with touchdown protocols do you have your temp decrease per cylcle all the way or does it stop at a set temp (hope this makes sense). Thank you vey much
with a normal template and primer control, this will test if your reaction conditions are working well, a positive control if you will, ideally it should be bisulfite DNA with a tried and tested primer set, anyone will do. Another problem that may arise is your thermocycler might need recalibrating and a service, the positive control will give indication of this and/or if your reagents are off.
As for touchdown, it's usually to a set temperature somewhere around the calculated Tm of your primer set, I set it such that it touches down to two degrees below the Tm,
I have been doing some methylation specific PCR as well, and have found that i have a much higher rate of allele drop out. I use RFLP to check for this after the PCR... perhaps you can check this in a PCR that does work - allele drop out has been a major cause for the random-ness of PCR in my experience
Thanks for the reply guys its been very helpful. Nick I have a positive control, methylated genomic DNA and while it does work for the most part the strenght of my bands tend to vary even though I'm using the same conditions.
I also had my tchdwn drop all the way down through 40cycles. I was wondering if anyone knows how to have the thermocycler level off, this is with GeneAmp 9700. I've looked at manual and all it does is decrease continuosly with every cycle. I am going to call ABI but I guess it'll have to wait for tomorrow in the mean time if anyone knows how to please enlighten me.
Jan I'm curious what enzymes you use for your RFLP? I am also wondering at what point to you characterize your samples as having an allelle dropout.
Thanks once again guys, words cannot show my appreciation.
The variation you are seeing could well be variation in starting template. How many cycles are you doing? if it's a low number this is probably why. Have you tried performing each PCR in duplicate or triplicate? if there is variations between the replicates then there is something awry.
for the touchdown on the 9700 you can set by how much the Tm decreases after each cycle, so from that, and the number of cycles you are using you can set the decrement so by the final cycle you would have reached your target Tm level. I hope that makes sense......
Thanks for the suggestions nick, I am doing my PCR at 40 cycles. I haven't tried my experiments in duplicates and that's what I'm going to do.
With the 9700 I set my annealing slightly above my primers Tm. The thing is with 40 cycles it decreases 1 degree with every cycle.
I think it just hit me, so let me run this by you. I need to set the temp change to decrease in such a way that at the end of my 40 cycles I hit my target Tm.
40 cycles should give a decent product.
lets see if you get a consistent result when performing duplicate reactions.
And yes, the touch down should end at a Tm comparable to the calculated Tm of your primers, I fear your method would topuchdown to a Tm of about 20-30C which is primer dimer territory!!