I'm trying to linearize a non-commercial vector for some cloning. We have primers that back up to one another, so when it amplifies outwards it opens up the plasmid. Or, at least, it did.
Without changing anything this reaction suddenly started producing smears. I've re-diluted both DNA and primers from original stocks that have been un-touched. One primer is pretty GC rich so while I was using a GC Phusion Polymerase I switched to an enzyme that hadn't produced my product but had at least produced a distinct band with no improvement.
The first picture was from figuring out the reaction conditions. Mix is pretty standard, mostly 50uL rxns with 2X buffers already containing dNTPs and enzyme and 2.5uL 10uM primers. My product is 5kb so there was definitely mis-priming but gel-purifying wasn't proving to be a problem.
I moved on to trying to test increasing annealing temperatures to make it more strict but immediately ran into an issue with even the program that had initially worked.
Second image is an example of what I'm currently getting, running the original program on the thermocycler. Not that it matters but in each lane I had re-diluted the primers and template DNA in two different nuclease-free waters.
Not included is an image of me running it with a different enzyme, which produced the same smears.
Third image is running a PCR on same template that worked (first two lanes) followed by this meddlesome PCR producing the same smears as always. The lanes represent running in two different thermocyclers, so the "good" PCR worked in both while my PCR worked in neither.
I'm probably going to forge forward and try designing new primers, but as I need this to open up the vector in a particular spot and a nearby region is so GC rich it would be great if I could figure this out as there aren't a ton of options there.
Has anyone run into similar issues?
Hi to you all.
One thing I keep saying here at RG is, please, send us a reply in every single forum question placed, letting us now If you solved your problem or not based on our discussion. It would help all others in a deeper way.
All the best.
what does your no template control look like.This sounds like post pcr contamination to me where great over amplification causes the product to anneal with other product molecules and produce a concatenation smear
Reduce the amount of template and try again. Seems like your template concentration is too high therefore it produces smear.
Thank you all so much! The template is 50ng in a 50uL reaction so I will try reducing it all the way to 10ng and work up from there if needed. Any idea why this would have happened suddenly when the initial reactions worked?
Goran, that is exactly why. I can't go into much detail but I need it to be right in between two genes and the intergenic region is extremely GC rich (80%+) so we don't have a whole lot of leeway to tinker with things.
This might be as a result of contamination, you should try using 2.5% DMSO. This might work.
Seems DNA concentration too high. And also check on the gel for degrafation. If working with the same DNA after a long time, dilute DNA.
Hi, Emerson.
In my opinion, you may be with two problems at the same time. Accumulation of amplicons contaminating your mix components and the high GC content. So, I would do one of three steps:
1 - Take a whole new amplification material (all reagents from another lab where you did not work with your amplicons or buy a new set and prepare everything in a lab outside your working areas) and re-test with a smaller plasmidial DNA amount (ranging from 100pg - 10ng of plasmidial DNA (re-extracted in another area and from another stock (sample and extraction kit);
2 - Do your amplification testing additives such as formamide, DMSO, glycerol in 3 concentrations each and a gradient PCR (ranging in 0.5ºC) for 3 - 5ºC as a total temp range. Still, when you resume your activity use a mastermix with UNG enzyme and partial dTTP replacement with dUTP (https://bitesizebio.com/4813/eliminate-pcr-amplicon-but-not-rtpcr-carry-over-with-ung/);
3 - design new primers just adding a few bases 5' up your actual primer. There is a paper about accumulation of amplicons as contaminating material which shows something close to your problem. Unfortunately, I just remember the author right now. His name was Han If I am remebering well.
Hope it helps you!
Check any of the following possibilities:
1. Too high template DNA: Reduce the volume of your template DNA in the mixture
2. Be sure that you try a new master mix and molecular grade water. Smears might sometimes be as a result of contaminations
3. Ensure you are not reusing your gel, at least for this purpose
Good luck
Firstly, you can use dilution of DNA (1:10 mean 1ul DNA,add 9ul dH2O)
and re use from this dilution 1ul DNA
secondary, change the annealing temperature to high 2 degree.
third, change the master mix
Good luck
Agree with Adriano Costa de Alcantara . I've had this happen to me before.
Try a gradient run, ive this issue before and usually changing the annealing temperature in a range of 0.5
Hi to you all.
One thing I keep saying here at RG is, please, send us a reply in every single forum question placed, letting us now If you solved your problem or not based on our discussion. It would help all others in a deeper way.
All the best.
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