No PCR product - troubleshooting PCR (Sep/21/2005 )
Hi everyone
I have been doing PCR regularly for HCV and HBV.
I usually extract DNA/RNA with Roche High Pure Viral Nucleic Acid Kit and never had problem with it. About a month ago I started to get no product in any of reactions.
I have changed every single reagent (Taq, dNTPs, Primers) and ordered new kit, tips, tubes and so on. still it is not working properly. rarely I get a very weak band, but this is one in every few time.
Does any one have any idea what is going wrong?
I suspect nuclease contamination! has any one experienced such problem?
Thanks
Shahin
Did you also check your DW, too?
Also it might be better to electrophrese your DNA/RNA sample before PCR.
Good luck anyway.
Do you have a positive control such as a plasmid DNA to rule out trouble i your DNA/RNA prep? At this point you need to figure out what is wrong. You seem to know where to look, but just need a plan how to get there.
Also it might be better to electrophrese your DNA/RNA sample before PCR.
Good luck anyway.
Thanks for your input. Well, I also used new DW as well as DEPC treated one.
Extraction should be fine as I did send a sample DNA (with no product in PCR in our lab) to my friend in another lab and the PCR worked fine there!?
Thanks for the tip. I don't have a Pos Ctrl like plasmid or so, but I did split the extracted DNA from Pos Ctrl and send half to another lab which came out fine though didn't work in our lab.
I have tried every thing I could think of and still nothing! I have been doing this for months on a regular basis and just suddenly nothing works!
have you cleaned out your pipettors? many have barrels that can be autoclaved?
if you are not using filter-tips, this could be a reason
I do not use filtered tips for my PCR, so every now and then I clean out the barrels with 70% etoh and a q-tip. I have not had nuclease trouble.
is your thermal cycler functioning properly?
I am assuming some sort of issue not related to template integrity somewhere (nuclease or equipment malfunction) since another lab got it to work
have you ordered entirely new synthesized primers, or just used different aliquots? your primer aliquots could be degrading in the freezer if it's been a while
wow, what a PIA; I hope you find the source
Try going over to the other lab and figure out what is different. I would say start from scratch and check out the other labs pcr protocol and follow it exactly. Unless you give me all the details of your protocol, it will be difficult to figure out hwat you are doing wrong. I doubt nuclease contamination is an issue. Primers do go bad when stored at a low concentration of extended periods. Everything else should be the same. Check that you are adding pcr buffer, mgcl2, dntps, primers, taq and that the pcr machine is set and working properly. Try 3-5 degrees annealing temp below your primers Tm. If your pcr machine heats from the bottom, try adding mineral oil to the top of the reaction to prevent evaporation to the lid. Just a start.....
Thanks for the points.
Just to clear, I use filter tips. by the time problem started I changed all the tip boxes with new sealed ones.
I am sure, I am following the protocol I wrote last year that was working fine every time, I even checkmark reagents after addition.
I use Min Oil and changed that one too!
The only thing I have not been able to check is the PCR Machine which we don't have any equipment to do so.
Does any one have a clue for a pilot test to check the PCR Machine before I send it out for repair or ask the technician to come and check it?
thx.
Do all the prep you normally do on the sample (combine Taq, primers, template, etc.), but at 2x volume. Split the PCR in half, and run one tube in your machine, and the other in the other lab's machine. If the sample run in their machine works, and the one in your machine doesn't, it's a problem with your machine.
clever, clever, homebrew....