Designing a touch down PCR

[saranyah]

Hello,

I'm having a trouble with primer dimers. How to design a touch down pcr? Or is hot start PCR better than that of a touch down?

[butters]

have you tried adjusting your PCR primer concentration and annealing temperature?

[saranyah]

I tried all sorts of changes in annealing temperature and concentration sir. I designed a simple primer without restriction site. And I got the gene amplified. But from the purified PCR product again I tried to use the old primers and I'm not getting the result.

[butters]

old primers? Do you mean amplifying the same PCR product with the same primer? If yes, most likely you didn't dilute your PCR product (some ppl use 1ul, if that didn't work you could dilute your PCR product to 1:10 dilution). Extremely too many DNA in template may inhibit if not retard your PCR reaction.

WAIT, what is the size of your PCR product?

If you are using another set of primer then it will be another thing.

[kan_das010]

What type of PCR are u using Now? I mean Touch down or Hot start or Normal PCR. See, If u r using PCR Product as a template for Next PCR, U have dilute the PCR Product if it is more concentration. Since u have purified the Product, once u can Check the concentration and use around 10ng DNA.

[saranyah]

The product length is 1.6Kbp. And the primers which has the restriction sites are not working.

[butters]

Saranyah, try google amplifying long PCR products/fragment. You would have lot of option there. In general I would look at the amplification duration (what is your current duration?), changes to the cycles in term of temp and duration, and make sure you have sufficient template to start with. Some ppl find that using a different type of taq polymerase help and is your template GC rich? cause that would create another problem.

[saranyah]

Please advice me on Fail Safe PCR system.....