Long distance (16 kb) PCR problem - (Dec/17/2005 )

We go up to 4 kb (and recently succesfully about 6 kb) on HIV RNA with a lot of secondary structure with Superscript III (not II) @ 55°C. We use the one step high fideliy kit, it works very good for us.

Apart from this, trial and error I'm afraid...

Hi every one:

When I try to amplify the partial fragment of this 16kb Desired gene, accurately, the forward half of this long fagment ( 9.5kb), I succefully amplified it with a final volume 10ul rxns system, but now I can not repeat it, even at the same condition and rxns system. I have repeated it 3 times, one time, I enlagre the Rxns volume to 50ul, all components are corespondently enlarged pro rata, how terrible and frustrated.

Any suggestion is apprecaited!