I am doing a PCR of my vector. I expect the PCR product to be of 5200bp. The PCR of the vector works only with 2ug DNA and Phusion Polymerase. It does not work at all with lower concentration of DNA such as 20ng, 50ng or even 100ng. I also tried to check the template vector (total: 6500bp) by just running it on gel. I can the difference between the two. But why only higher concentration of DNA is my question?
I doubt that what you see in the PCR is an amplification fragment. It might be denaturated template DNA. 2000 ng is an enormous amount of template. Also the size of the product you want to have is very big. You should probably try to amplify your region of interest by several separate PCR reactions and then assemble the fragments. If the PCRs don't work with usual amounts of template DNA something is most likely wrong with your PCR setup.
Regards, Christian
Why do you want to use such a large fragment of your vector? I would advise just carrying out a maxi prep and digesting the vector with suitable restriction enzymes, or if you need different ends then introduce some new restrictions sites by site directed mutagenesis and then go from there!
I agree with Christian that you are not likely to be seeming an PCR product, just the back bone.
If you run an agarose gel of your vector (un cut) how many bands do you see?
Thank you. @ Simon Cass: When I run the uncut vector, I see a smear but also a band lower at around 4500-5000bp. So I assumed it to be the supercoiled compact DNA so it ran fast. But I repeated it twice and I get the same result. I will try to follow what you said.
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