TA cloning system

[Andre-Denis G Wright]

(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 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.

: Usual disclaimers
: Cheers,
: DAJ

: 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!!!!!