Currently trying to amplify my 15kb plasmid with specific primers I designed for the desired ends of the linearized fragment. I'm currently using Phusion polymerase by NEB, and using the suggested cycling parameters and reaction setup. I add 2ng of my template, I use 15min extension time, and have used temperature gradient for the annealing temperature, but nothing has worked.
Below is a typical agarose gel of my PCR product. Right-most lane is the template running at the band size I expect. The other lanes are my various PCR products, and is running very close to the wells of the gel.
Can anyone tell me why the products are running so high? And any troubleshooting methods you can suggest for me to get the correct PCR product size?
Maybe you can try running a lower concentration/volume of pcr product for your gel electrophoresis
Maybe you need to change your extension time. NEB suggests 15-30s for 1kb extension, so for 15kb you only need 7-8 min of extension time.
Reduce the amplification cycle time to half and try changing the template concentration, reduce it too.
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