Dear colleaques

I have a problem with cloning of my PCR product (829 bp) into pET101 TOPO vector (5753 bp) by Invitrogen™ Champion™ pET101 Directional TOPO™ Expression Kit. I performed a PCR reaction to obtain my product, which I purified from the gel. The forward primer contains the CACC sequence as recommended. For PCR reaction I use Phusion Plus DNA polymerase whitch generates blunt ends. I got strong band with corect size and purified my DNA by Nucleospin kit. The concentration of my PCR product was 35,2 ng/ul.

For cloning reaction I follow the instructions in manual. It is important to use 0,5:1 to 2:1 molar ratio of PCR product:TOPO vector. So if I used 1 ul of vector (15-20 ng) I dilute my PCR product to concentration 3,52 ng/ul and add 0,82 ul of them into the cloning reaction (molar ratio 1:1) and incubated for 5 min (first time and 20 min second time with the same results). As control I used reaction only with vector (without PCR product). With reactions I transform One Shot TOP10 Chemically Competent E. coli and incubated on the agar plates with ampicilin (100ug/ml) owernight at 37°C. But the results were the same on both plates, where I got hundreds of colonies.

Then I took some 20 colonies and used them as templat in control PCR wth the same primers which I used at the beginning of the process. Reasults were negativ. This was also confirmed by restriction analysis after DNA isolation (miniprep).

Where could be mistake? Thank you very much for any advice.