pCDNA3 Cloning Difficulties

[Paul Edward Kowalski]

Regarding the occaisional "unclonability" of repetitive sequences in 
pCDNA3...
Very similar story : I'm using a 2.4 kb cDNA with about 400bp of sequence 
at the 5' end from a HERV-H element (a human endogenous retrovirus), and 
for the life of me, I couldn't get a simple EcoRI ligation into pCDNA3 to 
work! The same cDNA with the HERV sequences removed worked fine. I was 
using DH5a. Then I had the bright idea to transform the ligations into a 
different host strain- I used XL2-Blue and STBL2. Both worked, lo and 
behold, but by far the greater efficiency was using the XL2's. So the 
take home message- avoid DH5a when using pCDNA and repetitive sequences!

Paul Kowalski
Terry Fox Lab, BC Cancer Agency
Medical Genetics, UBC