I am trying to amplify a 6 kb fargment from a plasmid of size 6.9 Kb. I am getting my desired band alongwith some background shear. Could anyone tell me how to solve the problem. I am attaching a pic of the same for better understanding. The PCR conditions are: MgCl2 25 mM (0.5 uL), dNTP 10 mM (0.2 uL), primers FP and RP 10 pM (1 uL), hot start buffer 1 uL and Hot start taq (0.1 uL) for 10 uL reaction. Kindly suggest on what exact improvement to be carried out to improve desired band quality (on top) and remove shearing effect.
I would start by varying MgCl2 and, especially, dNTP concentration, which seems a bit too high.
Also try to increase your annealing temp by 1 degrees. I would suggest to do this 3 to 4 more times and each time increas by 1C compared to the previous repication. That would also help for avoiding non-specific binding.
I would also like to understand whether the GC content of the sequence is higher or normal and vary reaction by using DMSO (0.5-1%) if GC content is high in primer and the whole sequence. You can optimize the annealing temperature by setting up a gradient reaction with 10 temperature rise. What polymerase are you using and sometimes for large PCR products high efficient polymerases such as Phusion or pfu turbo polymerases work efficiently.
I am using hot start taq. The GC content of primers are are nearly 50%. I have actually carried out gradient from 64-68 degrees C.
Diptarka, check the section "smeared bands" on the link I've attached. It might help you.
http://www.bio-rad.com/pt-br/applications-technologies/pcr-troubleshooting#gel3
Hi Diptarka, just a few things.
First, I agree you should improve reaction conditions (MgCl2 seems high to me) and PCR annealing temperature by using gradient if you can.
Second, how much product (ul) did you load in your gel? it seems to me that you are over loading (adding too much DNA) the gel and that why you get those smears or background. Keep in mind that all PCR reactions produce non-specific products in a very low amount and then, if you add too much DNA you see them in the gel.
Good luck!
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