商品编号


E1201S-20 reactions



品牌


纽英伦(NEB)



公司


New England Biolabs



公司分类


PCR, qPCR & Amplification Technologies




20 reactions

商品信息

Description:
The Quick Blunting Kit is used to convert DNA with incompat
IBL
e 5? or 3? overhangs to 5? phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors. DNA is blunted using T4 DNA polymerase (
NEB
#M0203) which has both 3? →?5? exonuclease activity and 5? → 3? polymerase activity. T4?Polynucleotide Kinase (
NEB
#M0201) is included in the enzyme mix for phosphorylation of the 5? ends of blunt-ended DNA for subsequent ligation into a cloning vector. This kit is optimized for blunting up to 5 ?g of DNA in a single reaction. To learn more about how to identify what type of overhang you have, visit this video tutorial.
Blunt Enzyme Mix supplied in: 100 mM KCl, 10 mM Tris-HCl(pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% Glycerol.
1X Blunting Buffer:

100 mM Tris-HCl
50 mM NaCl
10 mM MgCl
2

0.025% Triton X-100
5 mM dithiothreitol
pH 7.5 at 25°C
Highlights
Fast — Restriction enzyme digested DNA is blunted in less than 30 minutes
Convenient — Reactions are performed at room temperature in a ready-to-use mix
Flex
IBL
e —Suitable for restriction enzyme digested DNA, sheared or
NEB
ulized DNA or PCR product
Kit Components
The following reagents are supplied with this product:
Store at (°C)
Concentration
10X Blunting Buffer
-20
10X
Deoxynucleotide Solution Mix?(1?mM)
-20
1 mM
Blunting Enzyme Mix
-20
20 reactions
Notes:
PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in
NEB
uffers 1,2,3 and 4, as well as BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in
NEB
uffer 1 where the total yield is about 50% of optimum.ATP is not necessary for T4 Polynucleotide Kinase activity in the kit. The dATP and dTTP in the dNTP mix act as phosphate donors.The blunted reaction?must be purified prior to phosphatase treatment by?using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.