How to eliminate primer dimer in MSP - (Jan/04/2006 )

Lisa,

these purifications can be made by the oligo synthesis company PAGE is more is expensive than cartridge which is more expensive than desalting.

For conventional PCR I only desalt, but for sequencing I cartridge purify, as MSP requires 3'ends to have perfect complimentarity, there is no harm in cartridge purifying your primers.

As for spec-ing your DNA......there is so little of it, and it's almost a DNA/RNA hybrid, the readings would almost be meaningless......the best way to check you have bisulfite converted DNA is with a PCR with primers you know that work.

good luck!

Nick

---

Hi,Nick,
You are always so helpful for us here.Now I can do methylation PCR from DNA of PB easily,but I can not see any bands from DNA of tissues.Could you please give me some suggestion?
I remembered I have seen this here before,but I can't find right now.
Thanks a lot!I appreciate .Lisa

---

Sorry, I'm thread-jacking but my problem is somewhat similar except I'm having problems with primer dimers in my BSP rxns. I have primers designed against both the unmodified and modified sequences and only have problems with the modified primers.

I've tried titrating input template as well as primer concentration but no help.

Thoughts?

thanks!

---

Hi Lisa,

I found that cell line DNA always performs better than tissue DNA although the same amount is used for modification, which means more cycles may be required to amplify modified DNA from tissue than DNA from cell lines.

---

Of course, primers for modified DNA tend to be problematic. What problem you have gotten? No amplification or smear? Please post your PCR conditions.

---

Thanks,pcrman.But I have already run 40 cycles,do you think I still need more?or can it help if I change the Tm and PCR protocol or DNA purity?I need amplify DNA from tissues,and I don't know how I can get it.
I sincerely wait here for an advice again!
Lisa

---

Ila -
A quick note. The 3' end of primers are not typically "frayed." Primers are synthesized 3' to 5' (opposite from the biological direction). So, the end of the primer which is problematic is the 5' end, not the 3' end. The usual errors are short primers or (much more rarely) deleted bases.

---

After the first 40 cycles, run another PCR of ~30 cylces using the first PCR product (with different diultions) as the template.

Using Hotstart taq such as JumpStart from Sigma helps a lot.

Try an annealing temperature of around 55C.

---

Thank you so much,My dear friends!I really,really appreciate.
Lisa

---

Hi everyone,

I have a similar problem. I have been doing a PCR on bisulfite treated DNA for years and it worked most of the time. Since end of 2005 it does not work at all!!!

Finally I sequenced and confirmed that I have strong primer dimer formation and NO PCR product!!! It is very frustating. Annealing T and Mg titrations did not help. When I read the other replys I realize that I am using too much primers - 1.0 uM and 20% DMSO. Can that be the problem? I am using Platinum Taq.

Please help, because I am getting totaly frustrated here...

---