long PCR of a DNA sequence with Alu repeats

John Ladasky jladasky at pmgm.Stanford.EDU
Wed Jul 22 08:40:04 EST 1998


In article <35b5c1c8.20774712 at news.fu-berlin.de>,
Thomas Burmeister <tbu at gmx.net> wrote:
>Hello newsgroup readers,
>I am trying to establish a long PCR with fragments approx. 4-13 kB in
>size. Up to now I do not get the expected gel band pattern (i.e. I get
>distinct  products, but not of the expected size).
>
> The amplified sequences contain multiple Alu repeats. Could this
>cause problems, leading to instability of amplified DNA (e.g. during
>the denaturation step in PCR ?)?
>
>Thanks for any response (please send a copy to: tbu at gmx.net)!
>
>Thomas Burmeister

Hello, Thomas,

	I regularly perform a 6 kB PCR that spans three Alu family repeats.
I get some weak side bands in the PCR, but nothing serious.  What are your
cycling conditions, and which reagents do you use?  I extend for 8 minutes
on every cycle, and I'm using the Stratagene Taq Extender system.  I also
found that getting my primers remade, HPLC-purified, and with the terminal
three nucleotides as phosphorothioates (S-oligos) greatly improved the 
yield and specificity of the PCR.  Good luck!


-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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