ligation/transformation: HELP ME PLEASE!!
salger at wap18.zi.biologie.uni-muenchen.de
Sun Mar 5 17:49:43 EST 1995
Heidi Moss (hmmoss at MAIL.MED.CORNELL.EDU) wrote:
: 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.
: -- 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
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 Salger phone : ++49 (0)89 5902 -502
Zoologisches Institut FAX : -450
AG MacWilliams e-mail: salger at zi.biologie.uni-muenchen.de
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