speI xbaI sublconing (dam methylation)

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Sun Dec 9 13:10:31 EST 2007


I DID have this (i.e. overlapping dam methylation) "problem" with  
XbaI. Of course it was totally my fault: not paying enough attention  
when I was designing primers. I couldn't understanding why I wasn't  
recovering clones for such a simple ligation... then I noticed that  
following digestion, the "vector" band was too big. When I looked  
more carefully, I realized I had designed my XbaI site with  
overlapping dam methylation. Every single thing I had prepped had the  
insert-- it's just digesting the plasmids (prepped out of XL-1 Blue)  
with XbaI would not release the insert! Needless to say, I've been  
pretty careful about this issue from then on!

Mike

On Dec 8, 2007, at 10:23 AM, Haviland, David L wrote:

> Mike:
>
> 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.
>
> David
>
>
> -----Original Message-----
> 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
> correct orientation.
> Good Luck,
> Mike
>
>>> "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
>>> the
>>>> 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
>>> enzymes
>>>> 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
>>    vice-versa.
>>
>>
>
>
>
>
>
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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