TA cloning system
Andre-Denis G Wright
awright at uoguelph.ca
Thu Feb 10 01:48:44 EST 1994
(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
: 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!!!!!
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