ligation/transformation: HELP ME PLEASE!!

Steven Loechel frosty at biobase.dk
Thu Mar 9 01:42:31 EST 1995


Heidi Moss (hmmoss at MAIL.MED.CORNELL.EDU) wrote:

: Would you believe, I am STILL having problems with the simplest cloning 
: exercise in the book??

: here is the story in brief:
: -- 2 HindIII fragments 1.1 and 2.7 --> genecleaned and ETOH precip. (others 
: in the lab have claimed problems with geneclean, either inhibitors 
: present for the ligation or transformation. I, personally, have never had 
: this problem, but the concensus is that it's the lot or it's old. I wanted 
: to be safe.)
: -- HindIII digested pBlue -->dephosphorylated-->genecleaned-->ETOH precip.
: -- ligated using brand-new BoeMannheim enzyme/buffer: 3:1 molar ratio 
: insert:vector, 10ul total (1ul ligase), 8 hours at 16 C.

: -- Transformed 3ul out of 10 (ligation rxn) into BOTH Sure2 cells and 
: XL1-Blue MRF': almost NO colonies (2-6) appear on the plate. PUC18 
: control looks great. 

Two likely possibilities:
1) You used too much alkaline phosphatase and damaged the ends of your
   vector.
2) You used short-wave UV light when you cut the bands out of the prep
   gel, damaging the DNA and making it uncloneable.

Frosty 




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