ligation/transformation: HELP ME PLEASE!!
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
2) You used short-wave UV light when you cut the bands out of the prep
gel, damaging the DNA and making it uncloneable.
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