Trying to design my first set of methylated primers. Ran a temperature gradient and think I’ve narrowed down a functional range of annealing temperatures across primer concentrations.
Now, I wanted to finally test efficiency across a few annealing more select temperatures. However, I’m realizing that in my labs qPCR protocols, most of them don’t carry an extension step, only the annealing and denature step.
My understanding is that since the templates and products are small (100bp) the copy is usually completed during the ramp up to the denature step. I’ll add that my Tms are approximately 64C, and my estimated annealing is consequently in the 55-61C range. We are using IQ sybr green super mix (iTAQ polymerase).
Just wanted to inquire if this was indeed the case, and if I should rerun my temperature gradient with an added extension step or just proceed with piloting.
On an agarose gel, I didn’t see any double banding across temperatures (suggesting high primer specificity?) albeit brighter bands were detected at specific temperatures, within 2C-6C of the Tm, so I wanted to run sample dilutions across a few degrees to maximize efficiency.
thanks for any help!
Most polymerases these days are capable of extending at 1000 bases per second and longer extension times are often used for great amplification of long products so the enzyme falls off in partial extension and the longer time allows another taq molecule to anneal in the half extended product and complete the amplificaion. Your product is very short so these thoughts do not apply. The lag times between temperatures should be plenty of time because the taq is extending even at the lowere annealing stage. One aspect that I would be careful of is that your product is the same size as primer dimer so make sure that you have a no template control to see what PD looks like in your amplification and when you get a product run a 2% agarose checker gel to make sure that you have a thin band of amplimer and not a diffuse smeary band of PD in the setup process
Efficient amplification of such short products does not need any extension time; it also needs no annealing time and no denaturing time (some denaturing time is needed for efficient separation of high-molecular template DNA during the first few cycles to avoid excessive re-annealing).
The critical part is just to actually reach the target temperatures in the whole volume of your PCR. If the volume is small, waiting for more than a few seconds is simply a waste of time. In rapid-cycle PCR, where the surface-to-volume ratio of the reaction tubes is typically large and the instrument allows steep heating and cooling rates, no waiting time at all is required in any step. In my personal experience, shorter cycling times typically increased the specificity of the amplification, too.
Adding to Pauls excellent answer I'd like to recommend using a 10-15% PAGE which separates these short products and primer dimers well:
4.5 % (w/v) Acrylamide
0.5 % (w/v) Bisacrylamide
0.08 % (w/v) APS
0.2 % (v/v) TEMED
in (1x)-TPE-Buffer
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