Not getting enough colony - (Apr/14/2009 )

This might be very silly problem to experts here, but annoying me for weeks, and i cant figure it out wheres the problem.
i am using invitrogen TOPO cloning kit, But not getting as much as colony i want. I guess, i need to optimize conditions. these are the conditions i used.
After PCR, I do Gel extraction of my band using qiagen kit, then i add Taq pol, Taq Buffer, dATP to get A overhang and keep it @ 72 degree for 10 minutes, then i do TA cloning using pcr4 TOP vector using 4 microlt of my sample, 1 microlt of vector, and 1 microlt of salt (according to invitrogen's ).
Then i use 2 microlt of this for transformation using TOP 10 cells, and proceed further.

What might be wrong? My insert is 246 bp (DNA from human skin), plasmid DNA is like 4 KB.

thanks

uv damage to the dna, impurities in the dna preparation. Can you try this without cutting out a band?

Did you screen the colonies you got? Did they contain your insert? Sometimes I got 2~5 colonies but I can still screen out the clone.

Why not use Taq Polymerase at the first place since your product is only 246bp, or use Stragtagene's EasyA HiFi, and add your PCR product to the TOPO vector immediately after the PCR rxn.
By the way, you might add too much insert, try 1ul or less.

Functional Screens on Apr 18 2009, 10:00 PM said: