Require help with Semi- quantitative PCR protocol - (Nov/09/2005 )
I have been trying to standardise PCR for rat CYP1B1 gene. I used primers that was already published and had standaridised the conditions.I even did a couple of runs with these conditions ad got beatiful bands at the appropriate position.
Recently the PCR just stopped working. I have re-ordered the enzymes, remade my cDNA and even changed the PCR machines...still no luck. Even control genes such as RPL19 and GAPDH dont work. I am tired and frustrated of getting plaing looking gel with only primer-dimer bands or no bands at all.
I am almost at the end of my rope and dont know what to do. Please help.
What enzyme are you using? I had a problem with one of those pre-mixed master mix kits where the lot to lot variability in sensitivity was like 100-fold.
The fact that all of your primer pairs are not functioning seems like it has to be the enzymes dNTPs etc, unless something happened that could have degraded or otherwise affected all of your primer stocks.
You did get a new batch of dNTPs right?? Bad dNTPs could also cause all of the problems you are seeing... (besides that if you used the same dNTPs to make your cDNA the cDNA synthesis would have been affected too...) If you didn't change the dNTPs, do that first...
HTH and good luck!
Thanks so much for the reply.
I am using Iscript cDNA synthesis kit from Biorad and Takara Taq polymerase from takara bio inc.
Recently I found that the freezer which I store my enzymes in (in the door) had been left open overnight and I did not want to use the enzymes anymore ( I had been having problems even before that...so the door being open incident might have been going on for a while!). So I threw the enzymes and oredered a new batch (BTW I moved the enzymes this time to a deep pocket in the freezer).
However I did not order new primers.
To figure out that my enzymes indeed were the problem I used a nighbouring labs Taq (which is ex Taq by the same company Vs rTaq which I use normally). I also borrowed their Gapdh primers. The PCR worked well for both my primers (RPL19) and theirs (GAPDH) when I did this. However the very next day when I ran a prep with my new enzymes (dNTP - aliquoted at -80C) It did not work.
Seriously I am baffled, as I crucially need the result for next weeks committee meeting
Can you think of something else??
degradation of primer or template?
So the template you used for the test PCR is the same template you used the next day with your enzymes? That would indicate the template is good...
The fact that your primer set worked with their enzyme may indicate the primers are okay too
This is strange to me, I also use Takara rTaq alot and I have found that the enzyme works well consistently, perhaps the lot of rTaq was improperly stored or otherwise affected by the shipping company?? I agree that the dNTPs stored in aliquots should be okay, but if you ordered new rTaq you should have gotten a new tube of dNTPs, did you use those? Which dNTPs were used in the test reactions with exTaq?
I would set up a reaction with the same master mix using your reagents, add the template to the master mix and then aliquot your primer sets (RPL19, GAPDH, and CYP1B1 and the other labs GAPDH primers) to each tube (or at least all the ones that you can PCR with the same program) then see if their primers still work, if their GAPDH works but your primers do not then I think you may have to order new primers (which will screw you up if you need it in a week so hopefully they all work or don't work together...)
Even better, borrow their exTaq enzyme for this set of reactions and figure out the mess after your meeting...
Actually I went back to my book and was looking when I aliquoted the dNTPs...if I had done it as soon as I got the kit it wouldnt be a problem..however if I did it after the freezer door incident they may have been spoilt even before I put them in -80.
I am going to do a PCR with the new dNTPs (I did the other PCR with the other labs dNTPs).
I know it is kind of really stupid but, I couldnt figure it out until I posted the second reply.
thank you for being my sounding board and all the advice.
Will let you know if it works...or else will come back with more questions if it doesnt...
Hope it works
It worked (yay... ). It was the dNTP s. .
However, the gene of interest is not amplifying....it is the same set of primers...now I will have to troubleshoot that. I used the same sample that I had as a positive control (RNA had been aliquoted and stored at -80). I am getting only a primer dimer band i.e. the band is about 100bp ( i think) at the very edge of the gel VS the 376bp band that is expected. Both housekeeping genes are amplyfying for the same sample ( band at expected size seen!).
I still think it would be worth ordering new primers
inconsistency is the primary symptom of primer degradation until they really fall apart, and it can take many weeks with data that starts out a little odd...then ever-more-confusing data; i.e. dimers, mispriming, multiple products formed, all sorts of wacky stuff...
primers are pretty inexpensive in terms of your other reagents and time for these experiments. I usually order two small batches of each primer I use at a time for my RT-qPCR. if a primer seems to be flaky for more than one experiment in a row, I've got some lyophilized back-up to reconstitute and keep the ball rolling. I lost almost 6 weeks the first time I had some primers degrade, and I mean I tested EVERYTHING else..it was a hard-learned lesson for me
I hope your 'fix' for this is an easy one, but unless your primers were really new, that's always a good culprit
It might be the pirmer. There are 2 things I want to know. Is it possible for the primers to degrade slowly. If so, will the stock rpimer degrade at the same rate as the dilute one ( of course considering the freezer fiasco, and the fact that I had both of them stored toghether ). Because, I had one experiment where I had superb results..and then it has been going downhill ever since until it came to a complete halt!. BUt the thing is the HK gene (RPL19) whose primers have undergone the same conditions as 1b1 seem to amplify...I am confused..yet, is it possible that there might be varying degrees of amplification or one primer design is hardier than the other??
Anyway, lets assume that I may not be able to use any of my.
Switching gears...what are your thoughts about using real time PCR primers (with amplicon size of 100 bp) in semi quantitative PCR.
the theory is the same, the method differs...I do not see why it would not work
I have heard that more dilute forms will degrade faster, but I am not sure why....maybe more chance of one or two little ambient nuclease molecules to crawl into the tube and degrade over time??
and yeah, it happens slowly; sometimes it works, sometimes it doesn't...I pulled my hair out and changed EVERYTHING else before I finally realized it was the primers when it happened to me...ordered up new primers and immediately got good results, though