topo ta cloning

as nospam at spam.dk
Sun Dec 26 16:46:55 EST 2004


Hi. I have a few basic questions regarding topo TA cloning

1) When I TA clone a taq produced PCR fragment into a given TA topo vector 
and subsequently sequence or colony screen the clones I usually find that 
most of the clones are in the same direction? But there should be random 
orientation of the fragments resulting in a 50/50 distribution?

2) After heatshocking the oneshot cells + ligation mix and adding SOC, how 
long time (minimum) does the mix need to shake at 37 degrees? As it is AMP 
selection I guess only around 10 min are needed, however the manual says one 
hour? Could I go down to 10 min, and how will this affect the cloning 
efficiency?
Thanks
AS
DK 





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