 |
|
 |
|
|
| Home >
Products >
DNA Modifying Enzymes and Cloning >
Cloning and Mutagenesis >
Quick Blunting™ Kit |
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
- 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
- Flexible —Suitable for restriction enzyme digested DNA, sheared or nebulized DNA or PCR product
Description:
The Quick Blunting Kit is used to convert DNA with incompatible 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 µg of DNA in a single reaction.
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 MgCl2
0.025% Triton X-100
5 mM dithiothreitol
pH 7.5 at 25°C
Applications:- Prepare sheared, nebulized or restriction enzyme digested DNA for blunt-ended ligation into a plasmid, cosmid, fosmid or BAC vector
- Prepare PCR products for efficient blunt-end cloning
Kit Components:
Blunting Buffer (10X)
Blunting Enzyme Mix
Deoxynucleotide Solution Mix (1 mM)
Storage Conditions
Storage Temperature:
-20°C
Notes
Usage 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 NEBuffers 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 NEBuffer 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.
FAQs
- Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit?
- Can I use regular ligase to ligate my blunted product?
- I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction?
- Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product?
- I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest?
- I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work?
- I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
The Quick Blunting Kit is tested with a pUC19 derived construct that is digested with ApaI and BamHI to produce 5´and 3´overhangs. This construct is blunted, ligated and transformed into NEB 5-alpha Competent E. coli (NEB #C2991) and spread on plates containing Xgal, IPTG and ampicillin. Blue colonies indicate precise 5´overhang fill-in and 3´overhang removal of the plasmid DNA.
Reagents Sold Separately
Deoxynucleotide Solution Mix
Companion Products
Quick Blunting™ and Quick Ligation™ Kits
Quick Ligation™ Kit
T4 DNA Ligase
|
|
|
 |
|
|
