pUC18/19 double digest with BamHI/SmaI

Michael Gregory Abel, University of Tennessee abel at UTKVX.UTCC.UTK.EDU
Mon Jun 19 13:15:17 EST 1995



On Mon, 19 Jun 1995, 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
> 
> 
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
						Abel at utkvx.utk.edu




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