cloning an AT rich gene
Posted 06 February 2012 - 08:17 AM
I am new to cloning and facing a big problem.
I am trying to clone an AT (80%) rich gene from Plasmodium.
I have PCR amplified the target gene, extracted the band from the gel, purified it with Qiagen Gel extraction kit.
I run it on a gel to estimate the concentration and ligated it into PGem plasmid using the PGem Teasy kit in a 1:1 molar ratio (insert to vector).
Transformation looks o.k.
I did colony PCR with insert primers and got a strange size band, 1.2kb instead of 550bp? what could this be?
the other problem is when I try to sequence it with SP6 and T7 primers I only get the vector sequence.
did the ligation not work? how could I test it?
Posted 06 February 2012 - 10:01 AM
how did the ligation plates look like? control vs ligation?
Posted 06 February 2012 - 10:46 AM
Posted 06 February 2012 - 12:05 PM
Also, you could just linearize your ligation product using a RE with only 1 site and just observe the length