Hello, I've been tasked with validating a 2kb region of DNA. A colleague designed primers with the F melting at 53.8 and the reverse at 59.7. I believe they used 54C which had multiple bands show up in their gel due to specificity issues. I am trying to optimize the reaction but don't know what temp would give the best results. Redesigning was my first thought but that's not an option. My plan now is to start at 56, see what I get if its still not specific enough than increase until I get clear bands. The problem however is this will be quite time consuming and will waste resources. Any input on next steps would be greatly appreciated.
Thank you
Redesigning is absolutely the best option. Also the cheapest.
You'll spend a load of time, energy and expensive reagents trying to optimise something that (by all accounts) is just...poorly designed.
Don't do that. Redesign a better pair of primers, save yourself the pain.
I agree with John Hildyard
However, you have one more option. Your PCR machine should have an option for gradient setup. So why not try a gradient PCR and find out the optimum Tm for the available primer pair? It works most of the time.
Hope this helps!
Best!
I've worked with a couple of horrible templates with really low GC content where redesigning truly was not an option. I was able to make most of them work by including LNA bases within the primer, which increases the effective primer Tm and allows it to remain annealed at a higher temperature. I can't find the reference I used but after doing some reading I decided to try 2 consecutive LNA bases placed 2nt in from the 5' end of the primer, eg: 5'NNXXNNN....NNN3' where X is the LNA. About half of the reactions worked well with temps around 2-3 degrees higher than the original primer. Primers containing LNA bases are expensive and there's no guarantee this will work, so I would only do this if there's a genuine sequence constraint which prevents you from designing better primers.
Primers are cheap, your time is valuable. If you can't get the primers to work better using a gradient to find a better annealing temp, then just scrap and redesign.
As a note, you can always increase the annealing temp by making the primers longer. Might be something to consider during your redo.
I agree that redesigning the primers to a higher annealing temperature is the best option. One other possibility is to run a sample with increasing amounts of dmso from 0 to 8%dmso which can often clean up a pcr reaction in much the same way as an annealing temperature gradient
Hi Ethan,
Higher temperatures is the only option if you are not planning to redesign primers. Usually specificity increases with higher temperatures. I guess a temperature between 57-60 will give the desired product. If it doesn't work add DMSO 1-5 % and see.
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