ligation/transformation: HELP ME PLEASE!!
hmmoss at MAIL.MED.CORNELL.EDU
Wed Mar 1 10:08:17 EST 1995
Would you believe, I am STILL having problems with the simplest cloning
exercise in the book?? I desperately need advice: in the past, I thought
my problem was trying to clone large inserts, but now that i've moved on
to a 1.1kb and a 2.7kb insert and my problems are still ever-present, I
am beginning to wonder... In the past, I have cloned into pBlue and bacteria
with great ease.
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.
-- Ran 1 ul of ligation on agarose gel: different
conformations/smear/bands above vector size (I was told this is good:
it shows that the ligation was successful and circularized; thus, it runs
based on different coiled-ness); little/none discrete insert/vector bands
-- 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.
-- I only mini-prepped the Sure 2 set (the XL1Blue will be done
tomorrow) and I obtained weird results: random, multiple bands. All
different for each clone. Perhaps I should hybridize to see if anything
is left from my original clone.
-- WHAT THE HECK IS THIS???? I AM FRUSTRATED BEYOND BELIEF! IT IS
SO DIFFICULT FOR ME TO PINPONT THE TROUBLE-SPOT. I FEEL I HAVE TRIED
EVERYTHING. I WANT TO GIVE UP.
Could it be a sequence problem?? After all, these are supposedly
5'/promoter clones that have an intron and exon present, based on initial
analysis. Could there be weird palindromes or repeats that the bacteria
doesn't like?? What part to you think is throwing me off?? Also, I just
did an experiment where I found that the gene is also slightly induced by
IFNB. I am suspecting a GAS element in the promoter, perhaps. It is a
far-fetched hunch, but could that cause a problem?? I am ignorant. I
don't know what to do now.
Many thanks for any help you can give me.
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