TA cloning vactor

byron byron_w14850 at yahoo.com
Tue Jun 27 18:08:21 EST 2006


I'm not sure what you mean about e-mailing you the Ta cloning vector,
but any vector can be used for TA cloning of PCr fragments, as long as
it has a blunt end cutting enzyme site, such as SmaI or EcoRV, for
example. What follows is my procedure for cloning PCR fragments into
pGEX using TA cloning:

T-tailing vector and T/A cloning

Digest 5-10 µg of DNA with blunt-end enzyme  (usually 10-20 µl of a
midiprep.)
e.g. SmaI: fermentas buffer  1x Tango Y+
or EcoRV: Fermentas buffer R+

Example of Digest:
10 µl vector
20 µl 10x Y+/Tango
5 µl SmaI
165 µl H20.
Digest for 37 degrees for 2-3 hrs.

Ethanol ppt. by adding 1/2 vol of NH4OAc and 2 vol of cold ethanol,
ppt. 1 hr to overnight in -20 freezer. Spin 15'' and remove
supe., wash in 500 µl 70% ethanol and spin 5'', pour off supe and
dry in rotovac, 10-15''.   Resuspend in 79 µl H20.

Tailing reaction
79 µl DNA
10 µl 10X PCR Buffer
8 µl 25 mM MgCl2
2 µl 100 mM dTTP
1 µl Taq polymerase

2 hrs., at 72 degrees (can use thermal cycler on manual control.)
Purify with mini-columns (Qiagen PCr purification kit), or ethanol ppt.
  final volume is 30 ul (columns) or 20 ul (etoh ppt.)

PCR Reaction: (for insert)
using Taq polymerase (non-proofreading) will result in A added to the
3' ends of amplified products. There is some preference of Taq
polymerase adding A to 3' end if C is present (not A), so if
possible, start primers with "G" at 5' end.  If proofreading
enzyme is used (like Pfu or Accuprime), then A can be added to the ends
of the amplified product using the above Tailing reaction recipe
(substituting dATP for dTTP), then repurify using Qiagen PCR
purification minicolumns.

Ligation (example)
1.5 µl vector
15.5 µl insert
2 µl 10x Ligase Buffer (fermentas)
1 µl (5 units) Ligase (Fermentas) [the regular one, not H.C.]

Mix, spin 1'' and incubate as follows: 1 hr at R.T., then 16
degrees overnight.

Transform 1 ul into XLI Blue and plate entire mixture on antibiotic
plates.



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