Hi, I put my PCR tubes in and forgot to press enter in order to start it. After 1 hour when I was expecting that it would be finished I went and checked that it was never started. Has anyone made the same mistake? If so what did you observe?
Hi Muhammad,
If you don't use a hot-start polymerase and keep the reaction for a long period of time, even at 4 degree Celsius dimer formation of your PCR oligonucleotides may occur and the oligonucleotides can be elongated if their 3'-OH end is annealed. This can change the sequence of your oligonucleotides, which would lead to false-priming and unspecific PCR products. If you consider that extension times of one minute per 1000 base pairs at 72 degree Celsius are normally applied, and take into account the Q10 temperature coefficient, the polymerase will work at a speed of around 8 base pairs per minute at 4 degree ...
Best,
Jochen
Your run should be finished now, so what did you observe?
Did you use a hot start polymerase?
nothing happened to my samples, it worked quite well...because i think pcr tubes were all the time (approx. 52minutes) on cold block at 6 degree celsius :)
If the samples are all kept chilled, then nothing should happen until the cycling is started. I have kept PCR samples on ice ready to go for several hours before beginning a program, and the results have been fine. I would not recommend doing that as a habit, nor would I recommend doing something like that overnight or longer, but the conditions you describe should not impact your PCR negatively.
Well you are lucky as the ramp was at 4 degree, otherwise chaos would have occurred. :)
Hi Muhammad,
If you don't use a hot-start polymerase and keep the reaction for a long period of time, even at 4 degree Celsius dimer formation of your PCR oligonucleotides may occur and the oligonucleotides can be elongated if their 3'-OH end is annealed. This can change the sequence of your oligonucleotides, which would lead to false-priming and unspecific PCR products. If you consider that extension times of one minute per 1000 base pairs at 72 degree Celsius are normally applied, and take into account the Q10 temperature coefficient, the polymerase will work at a speed of around 8 base pairs per minute at 4 degree ...
Best,
Jochen
I have set up PCRs using normal Taq polymerase (no hot start modification) ahead of time and frozen at -20 for half a day until the thermocycler in my lab was available. Thereafter I would give them a manual hot start at 95 deg C so that they would thaw and get heated rapidly to the denaturation temperature. The PCRs always ran as well as they would when I run them immediately after setting them up.
In my case, I forgot for about 3 hours, and then tried running it. It didn't work...Sigh...
Same here: Once I forgot my tubes in the cycling machine without starting it. Roughly an hour later I noticed and the PCR was unfortunately not working.
Add taq. pol into it and run the samples again. it will be definitely amplify.
I have done this and get good amplification.
Hi
Thank you Joachen for your very nice answer. I learned a lot form it. For you Muhammed, I think if the theromcycler block is cold enough your samples can still work. But I would I always add controls to my PCR and if any (odd) results show, I advice you to repeat your reaction.
Mohammed Farook, my previous colleagues provided you with great advises. What I want to share is similar story, when I added RNase to some samples for testing whether the the primers and probe can do the job, specially my targeted gene has NO splice junction. someone took my booking time by mistake, so I forced to wait and left my samples in -20 freezer. ALL my samples when disappeared, so I repeated my experiment. Mohammed, hopefully this will help you avoid similar mistakes. all the best
Hi Muhammad!
This depends for how long your samples were kept in the thermocycler. Your samples may work since the theromcycler block is cold enough to maintain your samples.
Good luck!
Hi Muhammad,
If you use hot start polymerase your pcr reactions should be fine as long as it was only for a few hours, the longer the time left out the more unreliable results you will get. Most current polymerases have now better fidelity and efficiency and seem to be a bit more temperature change resistant before activation. As others mentioned already, thermocyclers tend to be cold enough to protect your samples for a while. This happened recently to one of our students and the samples were ok when she actually run her plate again.
good luck
@Jochen C Meier
I do agree with your points
Hi Muhammad,
if your sample is not precious one, I will do a new reaction.
Hopefully nothing will happen...you can restart the reaction. Although there is a slim scope that primer dimers may have formed..
Good luck.
Nothing will happen, it may form some primer dimer, but still your PCR will work for sure.
Like everyone else says, I would just repeat it if your sample is not precious. If it is, then it depends on your goals. First, you should not have any other erroneous products from your template since your template is double stranded DNA and was never denatured so the primers could never anneal to it. If you used a hot start Taq, then chances are you will probably have negligible amounts of primer dimer. Even if you didn't use a hot start taq, you will likely have some primer dimers, potentially a lot if they keep amplifying during the reaction. This may only be an issue if you have very little template in the reaction so the primer dimer may out compete the desired product.
If your goal is just a positive or negative on template being present, then it shouldn't be an issue - use as is. Even if you want to isolate the product, you can do an agarose gel purification to remove undesired products. But on the whole, I don't think it's a big deal. For me it's only a loss in confidence. If I am doing something critical like sequencing where any negative impacts from it is something I can't predict, ideally I would not use it just to avoid any potential confounding variables from it. I like being very confident in my methods to come as close as I can to eliminating preparation technique being a variable in my results.
It happened with me once, how long your samples were kept in the PCR machine. I thank the PCR product will be ok.
As long as the DNA double strand is intact so long no issues with mis-priming and no chance of any non-specific amplification. Primer dimer is typically an issue that stays with how you have made the primers and the chances of primers to anneal also a homology dependent factor. At RT there are very little chance for primer dimer formation. Most of the t9ime we use the primers that are shipped over night at RT. If nothing happens then should not happen anytime within a short period. Longer incubation may possibly result a primer dimer issue. It may not be a bad idea if a simple PCR reaction could be run to clear the doubt.
@ Seth:
I disagree. The primers are single strand and hybridize with each other or themselves. Depending on the quality of the primer design, e.g. hairpins or simply primer dimers with 3'-OH annealed ends can lead to elongation of the primer and change the 3'-end binding sequence. This part of the primer is the most important one as the PCR amplification starts there, when you finally run the PCR in a cycler. As you can never rule out that such a thing happens, i.e. for reasons of reproducibility, I would rather discard a sample that was forgotten. If your probes are precious, you should not forget running the PCR...
Best,
Jochen
You can run the PCR and to prevent non specific priming you can first increase the melting time. But check the sample through sequencing. Non specific product can be eliminated when you will run gel, size of your interest will give you desired product.
I had reactions sit at room temp for an hour before running (it was a new machine and I didn't know you had to push start then confirm the start before the cycle would begin). The PCR worked fine. It was a set of routine genotyping samples with gDNA, standard lab primers, and no plans to do anything with the DNA after the PCR (just a gel, no cloning, digests etc).
(as a side note, letting cDNA synthesis reactions sit for an hour does not result in quality cDNA)
Was the lid heating on? If so, cast away the PCR. You get only difficulties. See Jochens comment.
Hi unless your samples are very precious in my experienece after 1 hour everything in all likely hood will still be fine
Indeed, I have even known people whose peltier blocks have broken and not been replaced and who thus are unable to perform a cold soak at the end of PCR let their tubes sit for hours and for simple ID purposes the PCR products have been fine
Most PCR reagents as mentioned are fine @ RT given that they are designed to be cyclically heated to above 90C for 30 cycles or so. What is more if you are using a hot start principle as mentioned nothing non specfifc would have happened after 1 hour and you template in any case is double strnaded and therefore not amenable to polymerisation (other than throough nicks in the DNA)
I wouldnt worry
To keep experimental results consistent I think it is best to start all over again; at least if you want publishable results... A bit of extra work, but you need a consistent method to get comparable data.
Lot of things ca go awry, for letting all things in the tube sit for a while and socialize. Depends on the PCR was meant for, what I mean is, If it is for the first time to check this or that, then by all means do all over again. Second if it was meant to check certain things and you already know from your earlier attempts the good and correct answer, then go ahead and finish the run, check if you get correct answer? I always teach that from a negative or incorrect test, one can derive good inference, for example when a PCR would be done with all possible right way you may get the same bad results when done in a wrong way, so one can figure out good and bad things for a single PCR results. Finally, to seek good and consistent results conditions has to be the same. primer-dimer, many non-specific products of different lengths. It can also lead into product of desired length.
We've made this mistake any number of times. If its a nice, robust PCR, it's usually fine. If not, the results are likely to be a mess. Nothing to lose by trying it, but I wouldn't trust any quantitative results.
Of course, it happens to everybody. Mostly dimers and non-specific bands will form. Nothing more than that.
Just like others have advised, you could decide to go ahead and complete the procedure, but the result will not be reliable. So it is advisable you repeat your test procedure.
once it happened to me too, no PCR was started for 1 hour and 15 min, I didn't use hot start polymerase, just Taq rec, but I started PCR anyway, and the next day I repeated an experiment just to check if I have differences, and there were no differences, except that "forgotten" PCR had weaker bands, but there possitions on a gel were the same, so in my case - nothing too bad happened.
Definitely worth trying. PCR mixtures tend to be very robust these days and you might find everything is just fine. If quantitative repeat but you would that in any case
Hi Muhammad!
Yes, I already experienced this and I had to repeat my PCR. You may get a positive PCR result even if you forget to start your cycling machine and start about one hour later. This depends the quality of your DNA (sample). I already tested my PCR this way by using a hot-start polymerase enzyme and it worked for some of the samples.
Good luck!
Hey,
I would rather start new reaction if the samples are important. If you keep the samples for some time (more than 30min./1h. even at 4°C) out of running PCR - without using hot-start polymerase - it could lead to unspecific PCR products and false priming.
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