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,
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°C
The single-digested plasmid is purified on a 1% agarose gel (overnight
– 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 – 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 – room temperature
The ligated product is loaded on a 1% agarose gel (overnight – 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.
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?
Thank you in advance,