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How do I PCR a DNA fragment with >200 CGG repeats?


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#1 2bornot2b

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Posted 15 January 2011 - 04:30 PM

Hi there,

I'm trying to amplify the triplet repeat region of a Fragile X individual who has >200 (CGG) repeats. This seems to be problematic, so any help with regards to primer design and PCR cycling parameters or PCR conditions would be much appreciated. What modifications would I need to make to a standard PCR reaction, and if possible, why would I make them... this would help me understand the rationale behind it all. I hope someone is able to help me figure this out.

Many thanks

#2 Maddie

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Posted 20 January 2011 - 07:39 AM

Hi there,

I'm trying to amplify the triplet repeat region of a Fragile X individual who has >200 (CGG) repeats. This seems to be problematic, so any help with regards to primer design and PCR cycling parameters or PCR conditions would be much appreciated. What modifications would I need to make to a standard PCR reaction, and if possible, why would I make them... this would help me understand the rationale behind it all. I hope someone is able to help me figure this out.

Many thanks


>200? Ouch!! I know that some Taq are optimized for GC rich regions. Have you looked into that?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#3 hobglobin

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Posted 20 January 2011 - 07:52 AM

Longer denaturation times in the cycles, some additives (DMSO, betaine (it is supposed that this is the main ingredient of the "Q-solution" from qiagen or "GC-RICH solution enhancer" from Roche)).

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.





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