PCR-screening of T-DNA inserted in Arabidopsis gene - (Jan/25/2013 )
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
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?...