No PCR product - troubleshooting PCR (Sep/21/2005 )
I though I would be telling the key to the mystry today! well I was wrong! Please take a moment and read the following and let me know if you have any idea what is going on. any advice or tips is highly appreciated as my brain has ran out of solutions!
Here is what I found.
1- I tried to extract DNA using TriPure/Trizol and use the extract in PCR. This way PCR works fine most of times, though still some times it doesn't work.
2-DNA extracted by the kit (High Pure Viral Nucleic Acid- Roche) is amplifiable in another lab but not in our lab (this is since 5 weeks ago, before it was fine).
3- Yesterday for the first time during the past 5 weeks I was able to amplify the target DNA extracted by the kit previously. the same extract (in the same tube) had been giving negative results before. however it didn't happen again today despite I tried to do exactly as yesterday.
4- this means that the reagents and the PCR machine working properly as DNA purified by TriPure can be amplified.
5- This problem is happening only for PCR. When I do RT-PCR it works fine (except for a couple of times during the past 5 weeks. Interesting! PCR is blocked but not RT-PCR. while the kit prep doesn't work for PCR, it is still fine as before for RT-PCR and I still purify RNA with it and every thing seems fine.
6- when I look back to my notes, there are some sporadic positive samples that have appeared Negative in PCR once in a 3 or 4 tests. so may be the unknow inhibitor has been around and know has contaminated every where.
Now I think may be an Inhibitor or blocking agent involved (like contamination with a chemical or so) which can block PCR when template is purified by kit but not (most of times) by TriPure!
looking forward to hear from you! thx
On templates where we've gotten sporatic results, we've found the PCRx Enhancer System from Invitrogen to be of enormous help. Maybe give something like that a shot?
Finally I found a clue!
I couldn't get the "PCR Enhancerx System" but used DMSO and nothing again happened.
I realized that RT-PCR reactions work fine and only PCR is not working. When I added RT buffer to PCR reaction (same as I do for RT-PCR) every thing seems to be working fine.
Apparently RT buffer increases the Mg concentration about 2mM. What I have to find is :
A) either there is something in my reactions that chelates/used Mg ions
or the PCR buffer despite the lable it says including MgCl2, doesn't carry MgCl2.
I am going to order some MgCl2 separately from PCR buffer and try to see what happens.
I'll let you know the results.
meanwhile thanks to all who responded.