2/3Kb PCR fragment - 16S/23S (May/13/2007 )
I m trying to amplify a 2/3Kb DNA fragment from bacteria DNA using universal prokaryotic primers. I m getting a very faint band plus a another smaller unspecific one. Not all of my samples are working. The Tm for my primers are 42 and 55 degrees. i m using an annealing temperature of 42. should I try higher or lower to get better results?HELP PLEASE (I am using a extension temperature of 72 for 3 min)
Can you extend the extension time?
Is the faint band is your desired band?
One of your primers is 42 degrees and you are using annealing temp of 42? Hmm... can you lower it down more? But again, you have another smaller unspecific band. Tough. Your band is pretty large.
How about MgCl2? How much are you using?
aside from timjim questions, what kind of polymerase are you using? With an extention time of 3min for 3kb product, I have a feeling you are using Taq. This is way to long a wait and Taq will not do for a product this long.
Although it is possible to amplfy PCR products upto 4kb with Taq, it becomes hard going and troublesome after around 1.3kb. (I personally don't use taq for anything pass 1kb) Please switch to a polymerase like KodHifi or Vent. You get high yeilds for porducts within the 2 - 5kb range. And your extention time is only about 1min to 1min30sec.
Aside from that, can you buy new primers? A primer with a tm 42 is hard going. If would be far better if you can buy new primers. Have their tm aimed at 60 celsius. The high tm, allows high annealing temperature, which gives better specificity, eliminating/minimising secondary band formation.
To remove the non-specific band, you have to increase the annealing temperature. But your tm42 primer, won't allow you. Given your primer's small size anything higher much higher then 42 celsius, and your primer would probably stop annealing to the template. (the melting curve gets steaper for smaller molecules)
To increase yeilds, you have to lower annealing temperature or increase MgCl2, KCl concentration. But all increases secondary product formation. A troublesome prospect as your secondary band is already apparent and will only get stronger.
although the Tm primer is 42 and 55, it doesn't means you have to do it at 42 or 55. furthermore like what Perneseblue have said, higher Tm is better, at 50 +. High fidelity Polymerase like Pfu, Pfx, phusion is very good for products around 5 kb, maybe you can try one. The temperature that worked best for me is 55 no matter what the primers Tm.