ligation/transformation: HELP ME PLEASE!!

Heidi Moss 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 
left.  
-- 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.
-- Heidi




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