| Product Information |
End-Repair Mix
(Low Concentration) |
| Part Number |
Y9140-LC-L |
| Price |
$250 |
| Concentration |
1 Rxn/µL |
| Unit Size |
200 Reactions |
Product Description:
The End-Repair Mix converts DNA containing damaged or incompatible 5′ - and/or 3′-protruding ends to 5′- phosphorylated, blunt-ended DNA. This low-concentration formulation of the End-Repair Mix is compatible with applications requiring <1 microgram of DNA to be prepared for blunt-end ligation. The conversion to blunt-ended DNA is accomplished by exploiting the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase (P7080). T4 Polynucleotide Kinase (Y9040) ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation by T4 DNA Ligase (L6030-HC).
Source of Protein
Purified from strains of E. coli that express the recombinant T4 DNA Polymerase, and T4 Polynucleotide Kinase genes, respectively.
Supplied in
100 mM KCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
0.1% Triton X-100
50% glycerol
pH 7.4 @ 25°C
Notes:
ATP is not required because the T4 Polynucleotide Kinase can utilize the deoxynucleotides (dATP and dTTP) used in the reaction as phosphate donors.
Usage Instructions:
- Purify DNA to be blunted, dissolve in TE buffer.
- Combine and mix the following components
in a sterile tube:
1-19 µL Purified DNA (up to 1.0 µg)
2.5 µL 10X End-Repair Buffer
2.5 µL 1 mM dNTP mix (N2060)
1-3 µL End-Repair Enzyme Mix
Sterile H2O to 25 µL
Total Volume: 25 µL
- Incubate room temperature (25°C) 30 minutes.
Inactivate End-Repair Enzyme by heat at 75°C
for 20 minutes.
- Ligation may be performed immediately using Enzymatics Rapid format T4 DNA Ligase (L6030-HC-L).
|
| Product Specification* |
| Storage Temperature |
-25 to -15°C |
| TEST: |
SPECIFICATION: |
| Purity (SDS-PAGE) |
>99% |
| 3′→ 5′ Nuclease |
Functional |
| 5′ Phosphorylation |
Functional |
| 5′→ 3′ DNA Synthesis |
Functional |
| DS Endonuclease |
10 µL = No conversion |
| E.coli DNA Contamination |
10 µL <10 copies |
Supplied With
B9140 (10X End-Repair Buffer)
N2060 (1 mM dNTPs)
10X End-Repair Buffer (B9140)
1 mM Tris-HCl
500 mM NaCl
100 mM MgCl2
50 mM DTT
0.25% Triton-X 100
pH 7.5 @ 25°C
Contamination Tests:
Purified free of contaminating endonucleases. In addition, >99% enzyme purity is analyzed by SDS-PAGE, and negligible E.coli genomic DNA is confirmed by qPCR.
Functional Assay
2µL of End-Repair Mix was added to 1ng of double restriction enzyme digested, dephosphorylated plasmid DNA in 1X reaction buffer containing 1mM dNTPs and incubated at 25°C for 30 minutes. Competent cells were transformed with the ligation mixure, plated onto LB/Amp/X-Gal plates and incubated overnight at 37°C. Control reactions consisting of End-Repair Mix without T4 DNA polymerase and/or T4 Polynucleotide Kinase were tested in parallel. The efficiency of the reaction was evaluated by comparing the number of blue and white colonies present in the End-Repair Mix plates to those of the control plates.
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