ligation/transformation: HELP ME PLEASE!!

[Klaus Salger]

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

Dear Heidi,
I had similar problems in the past. Once it turned out to be a
contamination in the ligase buffer. That seems to be unlikely in
your case. The other problem was a contamination in the EtBr-staining
solution (we stain the gels after running) or something else in the
fragment prep.
I solved the problem by just leaving out the fragment prep. When I
directly compared ligations with or without fragment prep I found that
the one with prep gave me very few clones (with wrong inserts) while
the one without prep looked really nice (lots of clones, >=50% with
the correct insert). And here's my recipe:

- cut out the fragment with RE + ScaI (at the same time in K-Glutamate-
  Buffer), heat inactivate REs
- cut vector with RE + dephosphorylate with Shrimp Alkaline Phosphatase
  (at the same time in KGB), heat inactivate RE + SAP
- run a test gel
- if digests are complete - take alliquots from the digests and ligate

ScaI blunt cuts within the Amp resistence. If it also cuts in your fragment
you could try another one like BglI (BglI-digest, blunt end, RE-digest).

If E.coli expresses your insert and doesn't like it, you could use
a low copy number strain (like Stratagene's ABLE) or a plasmid with
low copy number ori.

Hope this helps
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany