Home-Made TA cloning

martin LEACH leach at bu.edu
Wed Jul 19 09:27:23 EST 1995


I agree...Sma I or EcorV can be used to make a blunt end that can be tailed
upon incubation with Taq pol.

Interestingly, there is an enzyme (Xcm I)..possibly from New England
Biolabs.....

the digestion product from this is

.......xxx|xxx....
.......xx|xxx.....

or something like that...basically you can construct a vector that contains
this enzyme site and have it so that two T's are left overhanging upon
digestion...this is the basis of some vectors such as pDK101...and probably
those sold by the biotech companies...

If you request this vector on this newsgroup...there are still some people
that will freely distribute it to you....I was sent some but cannot send it
off since it is not originally mine...saves buying the digested T vector from
promega, clonetech, stratagene etc...

......ahh just found Xcm I....it is from New England Biolabs and the site is

...CCANNNNN|NNNNTGG...
...GGTNNNN|NNNNNACC...


i believe this method of T-vector construction was published in Biotechniques
a year or twoo ago...

good luck

Martin

.....          Martin Leach                Email:leach at bu.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
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 _/  |-/__==/  80 E. Concord St. (L603)
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s060965 at AIX1.UOTTAWA.CA wrote:
: I've been trying to think of a way to make my own TA cloning kit.  

: There doesn't seem to be any available restriction enzymes that will 
: produce the proper ends for TA cloning (although I'm sure Invitrogen must 
: have one in their bag of tricks).

: I've been thinking that if I ran PCR on a linearized plasmid 
: (Bluescript), and used primers that annealed to each end of the plasmid 
: but had one or two loose A's hanging over the edge it might work.

: Any ideas out there.

: Cris Martin



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