pUCm-T載體說明書
R04002 40T R04004 40T
R04003 100T R04005
200T
Components
|
COMPOSITIONS |
R04002 |
R04003 |
R04004 |
R04005 |
|
pUCm-T
Vector |
2μg |
10μg |
1μg |
10μg |
|
10×Ligation
Buffer |
|
|
100μl |
400μl |
|
50% PEG4000 |
|
|
100μl |
400μl |
|
T4
Ligase,5U/μl |
|
|
200U |
1000U |
|
Sterilized
ddH2O |
|
|
1ml |
1ml |
Intoduction
The T-Vector PCR Product Cloning Kit is suitable for
cloning of PCR products with additional A at 3’ end.The special ligation system
provided by the kit enables customs to finish ligation in 1-2 hours.
pUCm-T vector of our company is design for simplifying
cloning of PCR products.Many thermal stable DNA polymerase, PCR products
amplified by DNA polymerase such as Taq, Tth DNA produce additional A at 3’ edn
which could be easily ligated to T vector with additional T.
pUCm-T of our company is a kind of novel pUC derivative
T vector, the multiple restrict sits with most of them single site and adjusted
β-galactose reading frame
make it easily for screening target clone through blue and white
plaque.Specially designed two Pst I sites beside inserted fragments make it
easy for screening target clone by Pst I digestion, at same time inserted
framents also could be screened by cheap and efficient restrict enzymes such as
EcoR I and Hind III double digestion.Inserted fragments also could be sequenced
using universal primers M13 and T7 promoter primer.In vitro transcription could
be processed through site of T7 RNA polymerase promoter in pUCm-T.
Technical materials of our product are displayed at the
end of protocol,the complete sequence of our T vector are same as pUC19（GenBank Accession Number
M77789）except
difference at multiple cloning site.
Preparation of ligation reaction:
Purification of PCR products or not depends on quality
of amplified prduct. If PCR products are very specific,purification of PCR
products is not necessary,But if plasmids were used as template,it is necessary
to purify PCR product,because plasmid template could form white colony after
transformation.PCR products could be separated by agarose electrophoresis.
PCR products amplified by Taq、Tth、AmpliTaq、KlenTaq DNA polymerase
bear additional A at 3’end.Taq DNA polymerase co-amplify with Pfu、Pwo、Tli or Deep vent DNA
polymerase wich posses 3’—5’ exonuclease activity may bear a additional A at 3’end.
PCR products posses additional A at 3’end could be ligated to pUCm-T. PCR
products amplified by DNA polymerase with 3’—5’ exonuclease activity is blunt
end,cloning of this kind of fragments need to add additional A to blunt end.
MAP OF
pUCm-T VECTOR
pUCm-T|T載體|質粒
