topo XL cloning - (Jan/31/2006 )

I have some problems to clone a fragment of 3 Kb into a TOPO XL Vector. I used a quantity of fragment a 200 nm as suggested by manual, but I did't have positive colonies. What could be my problems? Have I to use a bigger quantity of fragment?

Did you use a proofreading polymerase do do your pcr? You may need to add regular Taq to make A overhangs.

after your PCR, add 1µl of regular taq and let 10' at 72°. Fragment is ready to use. (ansd need to be used quickly after A addition)

Indeed, first thing that comes to mind is the use of a proofreading polymerase.

Other than that: are you 100% sure about using the right antibiotic on your plates?

Any controls to check if your cells were still competent? (they should be, but you never know).

how quickly should you use the PCR fragment? I have had trouble with TOPO cloning too and wonder if loss of the A-tail is the problem. I did the PCR and then stored the PCR at -20. The following morning, I purified the PCR fragment and then did the ligation. Is 24 hours enough time for the A-tail to be lost?

I use sequenase, can it be a problem for the A-tail? How do I solve this problem? Should I change Taq ?