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PCR-screening of T-DNA inserted in Arabidopsis gene

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#1 Gwynbleidd



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Posted 25 January 2013 - 09:06 PM

Hi, I have a little understanding problem. I have gene from arabidopsis (homozygous) with inserted T-DNA. I have primers LEFT, RIGHT genomic (LG, RG) primers and left border (LB) primer of the T-DNA insertion. Now why I will not have pcr product when i will use LG and RG ? But i will have product when I will use RG and LB? T-DNA will stop elongating in first case? It would not go through T-DNA?

Like here for example http://www.pnas.org/....full.pdf page 2 fig 1A

Edited by Gwynbleidd, 25 January 2013 - 10:22 PM.

#2 bob1


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Posted 26 January 2013 - 12:26 PM

Could be any number of reasons - G/C content of the DNA? Too long for your cycling conditions? Tm not optimized? Mg2+ not optimal? incorrect primer?...

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