Trouble getting two large-sized bands - PCR (Oct/17/2006 )

I am attempting to verify a knock-out construction by the presence of an insert. If everything went as planned, I would have two fragments from my PCR--one about 4Kb and another about 6Kb. The end result was the 4Kb band, but no 6Kb band. I realize it is hard to bulk up the larger fragment, but this is the one I need to verify the KO.

I used Taq polymerase with the following 30 cycles:

94C 1min
52C 1min
72C 8min
Final extension: 72C 18 min

Can anyone make a suggestion as to how I can improve the larger band? If this has been discussed before, I would gladly accept a shove in the right direction! I just joined the board and haven't had time to search through all the previous posts.

Thanks in advance!

Correctly if I am wrong, but I don't think I have ever seen Taq amplify fragments more then 5kb.

You are really pushing it with the taq. Taq usually stops working at 4kb.

Could you not buy a primer or a pair of primers that would amplify across the junction between your insert and vector. It would be the cheapest and easiest (and certainly fastest) method I can think off.

But if you are hell bent on that 6kb fragment I would suggest

- lowering the extention temperature to 68 and increasing it further to compensate, (I have feeling this will not help.)

-using a proof reading enzyme with usually also have high processivity. that option is quiet expensive but would probably give the desired 6kb fragment.