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three fragments ligation problems - emergent help needed (Jun/23/2005 )

Hi, guys:

I have the following problems:

Vector: pBI121(12Kb, BamHI/SacI cut, CIP treated)
Fragment I: 3.7kb (BamHI/Xbal cut)
Fragment II: 3.1kb (Xbal/SacI cut)

Vector and the two fragments ligation, how can i get it success? Because I tried several times and failed to got the right clones, anyone who can give me some suggestions, thanks in advance for your help.





your ligation-efficiency with vectors of this size is very low anyway,
even with a single insert.
I'm working with 10-20 kb plasmids and I'm preparing my inserts by
combining them first in pBluescript and then moving them onto my
pRK404 or pVK100 or something else.
But I would not waste my time on trying to clone three fragments
at once with this size distribution.




I met the same problems as yours: Tripple ligation

My vector is 5.4 kb

inserts are 400bp and 1.8kb

Looking forward to others' suggestions on improving the efficiency.

Or I will design new primers and do my experiment again.....

But I don't have enough time anyway......sleep.gif