pUC18/19 double digest with BamHI/SmaI
M.C.Behrendt
or 19. I'm just wondering because the two sites on the MCS overlap by
one base, will this cause problems?
Mark
Michael Gregory Abel, University of Tennessee
Mark,
If you want to cut pUC18 or pUC19 with both BamHI and SmaI in a
double digest, you will probably have a hard time getting a good yield of
blunt(SmaI) and sticky(BamHI) ended linear vectors. The problem is that
the two enzymes have to "take turns" cutting at their respective
recognition sites. While BamHI can efficiently cut substrate DNA with as
little as two base pairs of flanking sequence (ie. BamHI cuts a DNA
substrate of sequence XGGATTCX (one base flanking recognition site) at an
efficiency of <10% but cuts a DNA substrate sequence XXGGATTCXX at an
efficiency of >90%), SmaI requires at least an overlap of 3 bases on each
side of the recognition sequence to perform >90% cutting.
Therefore, if both BamHI and SmaI are used to cut pUC19 (or 18)
in a double digest, the vectors which are cleaved by SmaI first and then
by BamHI would be completely digested:
XXXCCCGGGGATCCXXX----->XXXCCC GGGGATCCXXX (SmaI first)
BamHI cuts this floppy end >90% eff.
GGGGATCCXXX----->GGG GATCCXXX
However, if the vector substrate in the digest is attacked by
BamHI first in the reaction then there is not enough flanking sequence
for SmaI to efficiently cut when its "turn" comes around:
XXXCCCGGGGATCCXXX----->XXXCCCGGG GATCCXXX (BamHI first)
SmaI cuts this floppy end <10% eff.
XXXCCCGGG------->XXXCCC GGG
If you absolutely need to have an open vector with a blunt end and BamHI
end, I would suggest doing the digestion with the enzymes one at a time
with SmaI first.
All of the previous not withstanding, trying to do a double
digest with SmaI and BamHI may be tricky because the enzymes have very
different buffer and temperature requirements (BamHI requires a high salt
buffer and SmaI works best at 25oC (only has a half life of 10-15 min.
at 37oC). If you can find other sites to use in your cloning (I am
assuming), you will probably get better results in the long run.
Good luck,
Greg Abel
University of Tennessee
Dept. of Microbiology
Ab...@utkvx.utk.edu
Mikrobielle Genetik
If one can believe the studies undertaken in the BRL laberatory by Joe
Crouse and Dough Amorese, a double digestion with SmaI and BamHI will
cause no problems, digestion will complete.
Stephan
Colin Rasmussen
with BamHI and HincII (also a blunt-ended cutter).
bw...@mzdmza.zdv.uni-mainz.de
(M.C.Behrendt) wrote:
> Has anyone tried to do a double digest using BamHI and SmaI with pUC18
> or 19. I'm just wondering because the two sites on the MCS overlap by
> one base, will this cause problems?
>
> Mark
Hello,
You can take a puc-vector with an BamHI Insert in it.
Then you can cut with SmaI (in the MCS) and then with BamHI. This should
cut the distant BamHI-site in the MCS. If the Bam-site near the Sma-site
is cut does not matter. You can then separate Insert and Bam-Sma-cut
vector and insert by electrophoresis and elute the vector.
good luck,
Bernd Weil