Failed TA cloning with Fusion PCR product! - I need help with TA cloning of an Overlap PCR product (Oct/20/2009 )
I have successfully overlapped 2 PCR products with a Fusion PCR
The fragments come from two different organisms. I personally amplified the two genes of interest, then fused them together.
One is 750bp the other is 1000bp, and after the fusion i have a fragment of 1750bp. On the agarose gel, it's one clear band.
I want to ligate this DNA fragment into a vector then transform into E. coli.
I have tried everything
My vector is the pET-Blue, and it works with TA cloning, so no need for RE ligation.
I worked with both Taq polymerase and Hi-Fi Expand polymerase, which are known to add hanging A's at the end of the fragment.
I have tried to ligate the Fusion PCR product into the PET-Blue vector, then transform into Nova Blue cells but that didn't work.
I repeated the ligation/transformation step over 6 times, and got all in all 10 white clones, who all didn't have my chimeric gene.
I thought maybe the fused PCR product wasn't enough, since the band was too small, and OD at 260 was too low.
so I reamplified my Fusion PCR product with Taq pol, and tried to ligate my new product but that didn't work either.
What could the problem be?
Why can't i ligate my fusion PCR product?
The funny thing is, i cloned the 1000bp fragment 3 weeks ago into the same vector and transformed into the Nova Blue, (same protocol used) and that worked without any problems!
Any ideas or suggestions would be greatly appreciated!
We are using routinely T/A cloning kit from Promega.
T/A coning is known to be inefficient.
Therefore, we amplify the product to obtain clear band on the agarose gel.
Then we extract the DNA from gel, dry the entire eluate volume with the SpeedVac and add all the components for the ligation reaction.
After O.N ligation on 4C, we perform the transformation.
Therefore, prolonged incubation with high insert : vector ratio may solve the problem.