TA cloning system

txpljfg at UABCVSR.cvsr.uab.edu txpljfg at UABCVSR.cvsr.uab.edu
Thu Feb 10 12:47:04 EST 1994


The kit worked pretty well for us using either their competent cells or
our own cells through electroporation.  What I could not handle was the
godawful expense.  We didn't really need the cells, pcr controls,
ligation reagents etc.  Lately, we have been making our own T-vector
from pUC19 by cutting it with smaI and tailing it for three hours with
taq pol  at 75C in standard 1X PCR buffer (w/ 1.5mM MgCl2) containing
2mM dTTP.  Phenol extract it, ether extract it twice (hey..so I'm
paranoid), ppt it w/ ammonium acetate and isopropanol.  We have found
that our vector gives slightly higher backgrounds (probably due to
incomplete cutting).

We use color selection, and find that approx. 95% of the white colonies
contain inserts.  Most of the products we clone are between 100-300bp,
so often get light blue colonies that also contain inserts.

I think the kit is wonderful for labs that clone PCR products
occasionally.  But, for those of us who do mass cloning and creation of
libraries by PCR, the kit is just too expensive.  If they sold just the
vector, then it might be worthwhile to buy it if the price was right. 
However, rumor has it that ATCC has some type of vector that can be
used for TA cloning.  When (if) this becomes available, we will
certainly try it.


> (David Johnston) daj (daj at nhm.ic.ac.uk) wrote:
> : On 6 Feb 1994 08:41:45 -0000,
> :   Eric Yang writes:
> 
> : >Could anyone out there tell me something about the TA cloning system
> : >(Invitrogen) for cloning PCR products?  What are it's advantages and
> : >disadvantages?  Does it really work?
> : >
> : Yep, it sure does (for us anyway). The new version is better...

   >stuff deleted<
> 
> : David A. Johnston
> : Dept of Zoology, The Natural History Museum, Cromwell Road,
> : South Kensington, London SW7 5DB.
> : (tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)
> 
> 
> the kit sucks.....transformations must be done immediately after PCR
> amplification of else the A-overhangs will fall off the PCR product. As
> well, once you dissolve the lyophylized plasmid, it too will lose its T
> overhangs. We found in our lab that we got mis-matches which resulted in
> thousands of false positive white colonies. In addition, the competent
> cells are not the best. We tried everything we could and after 4
> months..NOTHING WORKED!!! Good Luck!!!!!
> 

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
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