how much lambda vector do I need to use

Ken Soderstrom soderstk at ucs.orst.edu
Fri Feb 14 20:51:26 EST 1997


Dear Cloners:

Soon I will begin construction of a cDNA library using Pharmacia's
"TimeSaver" cDNA synthesis kit and Stratagene's ZAPII vector.  

I've made a library with these kits before (using oligodT primers and
unfortunately am missing about 200 bases from the 5' end of my clone of
interest), now I'll make another library with random hexamers and, perhaps,
a bit of my sequence of interest as primers and screen again (I screened
the hell out of the first library).

My question for those with more experience is: how much vector do I need to
use?

Stratagene sells cut & CIPed vector in 10ug aliquots. Pharmacia's
"TimeSaver" cDNA synthesis kit recommends 3 test ligations using various
amounts of cDNA and 2ug of vector each, followed by a final, scaled-up
ligation based on test results - I don't think Pharmacia is limited by the
cost of vector - I am.  I'm also bound to use these kits.

Two ug of lambda per ligation seems excessive (although it did work
before).  I'd prefer to scale-down these recommended test ligations and
have plenty of vector for a final ligation or additional libraries.  

Does anyone have any thoughts on how to reduce vector use in this
situation?

Thanks in advance,
-- 
Ken Soderstrom
soderstk at ucs.orst.edu




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