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Designing a touch down PCR

Designing a touch down PCR - PCR - Polymerase Chain Reaction Forum

Designing a touch down PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-27-2012, 09:58 AM
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Question Designing a touch down PCR



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?
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Old 06-28-2012, 03:47 AM
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Default Re: Designing a touch down PCR

have you tried adjusting your PCR primer concentration and annealing temperature?
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Old 06-28-2012, 10:13 AM
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Default Re: Designing a touch down PCR

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.
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Old 06-29-2012, 03:45 AM
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Default Re: Designing a touch down PCR

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.
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Old 07-01-2012, 07:30 PM
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Default Re: Designing a touch down PCR

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.
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Old 07-02-2012, 07:11 AM
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Default Re: Designing a touch down PCR

The product length is 1.6Kbp. And the primers which has the restriction sites are not working.
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Old 07-03-2012, 08:27 AM
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Default Re: Designing a touch down PCR

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.
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Old 07-05-2012, 08:21 AM
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Default Re: Designing a touch down PCR

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