TA cloning system

[Paul N Hengen]

In article <2jclcc$b5h at nermal.cs.uoguelph.ca>
awright at uoguelph.ca (Andre-Denis G Wright) writes:

>(David Johnston) daj (daj at nhm.ic.ac.uk) wrote:

>:   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 than the 
>: original as it uses ampicillin selection rather than kanomycin so you 
>: don't have to make yet another type of plate. The only dissadvantages we 
>: have found are (1) copy number isn't particularly high (2) you get 
>: mountains of legal paperwork with the kit which you are suppossed to sign 
>: anmd return, swearing on your granny's grave that you won't give any of 
>: thre plasmid away etc..
>
>: Promega do a pGem T kit which does the same thing, works well in our 
>: hands, has a higher copy number, uses ampicillin selection and JM109 cells 
>: (our lab standard so no worries if the supplied competent cells run out - 
>: TA cloning uses INF alpha cells). Worth considering also.
>
> 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....

I've never used this kit or any others for TA cloning. Could you explain how
or why the bases are able to "fall" off?

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************