I have a problem to clone a PCR product into the pCR2.1 TOPO vector. The PCR product (300 bp) was synthesised with the Platinum taq DNA polymerase Hifi from Invitrogen. After the gel electrophoresis, the product was extracted with the Qiaex geletraction kit.
For the TOPO cloning, I used the following mix:
(TOPO TA cloning KIT with pCR 2.1-TOPO from Invitrogen)
purified PCR product: 1ul to 4ul (between 10ng and 100ng)
TOPO vector: 1ul
Salt solution: 1ul
add bidest water to 6ul
After 30 minutes of incubation at RT, I used 2ul to 4ul for the transformation into the TOP10 E.coli cells.
Result: A reduced number of clones (90 clones) where 2/3 of the clones were blue
I checked the transformation efficiency of the cells: OK
I made an A-tailing after gel extraction: no change
What I am doing wrong?