plasmid re-ligation problem

Wolfgang Schechinger hubahopp at gmx.de
Thu Aug 12 04:57:48 EST 2004


Dear Vagelis, 

BamHI in my experience sometimes is a bit nasty. Simply means, it doesn't
cut always to completeness. Your obeservation of self ligation probably
just means high background of not completely cut vector or sngle cuts which
are easily re-ligated.

My suggestion to overcome your problem is to add a third (or even fourth)
enzyme that cuts anywhere between the Xho I and BamHI sites. See the
compatibility table in the NEB catalogue. Will reduce background
significantly. And, of course, add some SAP to your vecor digest.

Best of luck and regards,

Wolfgang

PPS please tell us the outcome, we like feedback here in the Bionet!


At 11:48 11.08.2004 -0700, you wrote:
>Hello everyone,
>
>I am trying to construct a recombinant pSP189 vector that is going to
>be used for future site-specific adduct studies. In brief, the parent
>vector is digested with the XhoI and BamH I restriction enzymes (from
>NEB). Both enzymes are single cutters and produce incompatible sticky
>ends, and the distance between the two restriction sites is 114bp. We
>want to ligate back to the double-digested vector a modified,
>synthesized ds114mer.
>
>However, I encounter a problem during the cloning procedure; the
>double-digested vector re-ligates back to itself, even though the two
>ends are incompatible.
>
>The parent plasmid is digested separately with either the XhoI or BamH
>I enzymes according to the following protocol:
>
>-	10ug pSP189 plasmid (1ug/ul)
>-	10ul BamH I or NEB 2 Buffer
>-	1ul BSA
>-	100 NEB units of BamH I or XhoI
>-	up to 100ul with ddH2O
>-	2 hours, 37=B0C
>
>The single-digested plasmid is purified on a 1% agarose gel (overnight
>=96 40 volts).
>The enzymes appear to cut efficiently, when used separately, as is
>evident from direct DNA sequencing of the plasmid and also from the
>agarose gel photos
>
>The gel slices containing the single-cut DNA are excised from the gel
>and the DNA is purified with the QIAquick gel extraction kit (QIAGEN).
>Then, the second digestion is performed (same protocol) and the
>double-digested plasmid is purified again on a 1% agarose gel
>(overnight =96 40 volts). The plasmid DNA is recovered again with the
>QIAGEN kit and then it is used for the following ligation reaction:
>
>-	1ug double-digested plasmid
>-	1ul  (400 Cohesive End Ligation Units) T4 DNA ligase (NEB)
>-	5ul T4 DNA ligase buffer
>-	up to 20ul with ddH2O
>-	2 hours =96 room temperature
>
>The ligated product is loaded on a 1% agarose gel (overnight =96 40
>volts). The double-digested vector re-ligates proportionally to the
>amount of T4 DNA ligase. In fact, virtually all of it re-ligates in
>the presence of 4000 units of T4 ligase. Also, when the modified
>114mer is added to the ligation reaction in high concentrations, it
>tends to inhibit the re-circularisation of the vector.
>
>The same problem occurs even when I perform double digestions or when
>I do not purify the digested vector with agarose electrophoresis. I
>also dismissed the possibility of contamination in my ligation buffer
>or in the T4 ligase.
>=20
>My guess is that there is either a misdigestion problem during the
>second digestion or that the 114mer is indeed excised but not
>separated from the rest of the vector.
>
>Do you have any suggestions on how to overcome this problem?=20
>Thank you in advance,
>
>Liapis Evagelos
>PhD student,=20
>Biocentre
>Leicester University
>
>
Wolfgang Schechinger


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