Multiplex problems - (Jan/27/2011 )
I am trying to carry out this multiplex reaction using Allele specific primers. I dont think there are issues with primer design, cause I get bands and the primers are from this published paper(I can send the paper to anyone who is interested). For a heterozygote sample,I must get two bands one at ~250bp and the other at ~500bp. The Polymerase used in the study is not good at amplifying longer fragments, but the one I am using is, so at times I also get this added product of ~750bp.
My main aim was to get rid of the unspecific band, which I tried using higher annealing temperatures and lowering extension temperatures. Although, I got rid of the unspecific band (I slept over it for a while, cause I was doing other things), the reaction conditions which were working well then, have now also caused the disappearance of the ~500 bp band. So, I am getting only one band where I should get two and dont quite know what to do next, cause the changes I will now make, will take me back to where I started from (start getting unspecific band).
Basically, what I am looking for are ideas to strangle a good polymerase, so that is does produces a ~500bp product, but not ~750bp product.
Current annealing temperature is 63oC for 30secs (at 64, annealing is poor, gives no bands). Extension temp is 70 for 25 s (at 72, unspecific bands, 66, 68- no 500bp product). Extension time of 20 s does not give proper bands either.
Thanks for your help
Most polymerases polymerise at a rate of 1000bases/minute. Therefore, I would change the extension time to at least 35-40seconds. I hope this helps.
Did you check the primer sequences? Many articles contain errors. If the primers are OK and you use the published conditions unsuccesfully, then I'd try to contact the co-authors of the article to ask them if they ever had similar issues.
I guess you had tried on the DMSO, BSA or Betaine addictive, the increase buffer strength and/or Mg2+ concentration etc...... but it didn't work right?
Check the ramp rate of the thermal cycler used by the paper and compare with yours. I had worked on a multiplex PCR which will only work in a specific ramp rate.
p/s: no absolute guarantees I might able to help, but I do interested in the paper you are referring. Mind to pm me?
Thanks for you responses...
Maddie: I think the primer sequences are right. purely because I am getting bands in the right places. The published paper shows weak/ very weak bands. But I get a few unspecific bands as well. I think even if I contact the authors, they will say, use the polymerase we used and you should get what we got. I dont want to buy a polymerase for a single reaction.
Dimitris: I think I'll try doing what you have said.
Adrian: Actually, I have NOT used any PCR enhancers so far. Actually, since I started working here, I have not used any PCR enhancers, we use human DNA, so there is always a good PCR product. I shall look into the ramp rate. Anyways, see your pm... and let me know if you have something else to add..
After I had look into the paper, I feel that this is most probably due to the ramping rate as I suspected earlier: they also mentioned in page 7 "application to other thermal cyclers may require further optimization since different reaction vessels and heating/cooling methods can dramatically change the dynamics of a PCR.". They are using MJ thermalcycler which is a very old thermalcycler, and the "HS TAQUANT-OFF" in the PCR master mixed indicates that it is some sort of hot-start antibody/reagent.
As you had mentioned earlier, their polymerase is not good in amplifying large fragments. I try to check about the composition of their 10X taq buffer but I couldn't get. I suspect maybe you can try to increase your Taq Buffer to see if it helps in your reaction.
Hope this help.
Feedback on my PCR:
After three days and doing absolutely nothing.... different, rather not even setting up another PCR, I got the desired result.
Well, from the PCR I had set up before posting my problems here, there was one sample which i had not loaded onto the gel (too many samples for my gel). I ran it yesterday and Viola!!!! it had worked...
Yes, That makes it less reproducible... but I will work on your suggestions and see if I can make it work...
Adrian: Sorry, I had not seen your last post until today...