speI xbaI sublconing
Haviland, David L
(by David.L.Haviland from uth.tmc.edu)
Sat Dec 8 11:23:17 EST 2007
I won't argue with you but in my limited experience with XbaI and using various vectors in HB101, XL-1 blues, Top 10F's, and SURE cells, what problems I had in ligation were never chased to XbaI methylation. I dont' doubt what you are saying and perhaps in other bugs it would make a difference.
To me the compatible overhangs are what dogs this ligation, and I'd reach for the SAP in a heartbeat.
From: methods-bounces from oat.bio.indiana.edu on behalf of Michael J. Prigge
Sent: Fri 12/7/2007 7:19 PM
To: methods from magpie.bio.indiana.edu
Subject: speI xbaI sublconing
You didn't mention any problems with the digestion, but I thought I
would throw in this reminder, just in case. XbaI is susceptible to
dam methylation, so TCTAGAtc and gaTCTAGA will not be cut. If this
isn't the problem, I would try Tom's options, possibly screening
colonies by PCR with an insert primer and a vector primer for the
>> "V Arunachalam" <vadivelarunachalam from yahoo.com> skrev i melding
>> news:mailman.782.1196360538.23109.methods from net.bio.net...
>> > I am facing problems in cohesive subcloning a 500 bp insert from
>> > double digest of SpeI and XbaI onto a vector of 13.5 Kb size at
>> the same
>> > sites. Please suggest me is it correct strategy as these two
>> > generate similar ends though differ in recognition sites.
> Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu)
> Two options:
> 1) Ignore the problem and select one of the two orientations after the
> fact. About half should be in the orientation you want.
> 2) Add SpeI or XbaI to the ligation mix. This will force the ligation
> which recreates the SpeI or XbaI site to be cut, favoring the
> ligation of the vector SpeI site with the insert XbaI site, and
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