Fast DNA End Repair Kit
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This kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.
Detailní popis
The Thermo Scientific Fast DNA End Repair Kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.
All components of the kit contain premixed reagents to reduce pipetting steps and provide convenience. The End Repair Enzyme Mix contains an optimized mixture of T4 DNA Polymerase and Klenow Fragment to achieve highly effective blunting of fragmented DNA, and T4 Polynucleotide Kinase (PKN) for efficient phosphorylation of DNA ends. The 10X End Repair Reaction Mix contains an optimized reaction buffer, ATP, and dNTPs.
Samples such as fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated), restriction enzyme digested plasmid DNA, double stranded cDNA, and PCR products containing dA overhangs are all compatible with the kit.
Features
During the DNA end repair reaction, fragmented DNA is converted into blunt-end DNA containing a 5’-phosphate and 3’-hydroxyl groups. The 5’→3’ polymerase activity of the End Repair Enzyme Mix fills-in 5’ protruded DNA ends while 3’→5’ exonuclease activity removes 3’-overhangs. T4 PNK adds 5’-phosphates to ends of unphosphorylated DNA fragments, such as PCR products. (see Table 1 and Figure 1 below).
Highlights
- Efficient - blunting and phosphorylation of 0.5 to 5 µg DNA
- Fast – reaction is completed in 5 minutes
- Convenient – reaction components are premixed to reduce pipetting steps
Applications
- Blunting and phosphorylation of double-stranded DNA (nebulized DNA, sonicated DNA, restriction enzyme digested DNA, cDNA, PCR products)
Includes
- End Repair Enzyme Mix
- 10X End Repair Reaction Mix
| Activity | Enzyme providing the activity | Type of DNA ends repaired |
|---|---|---|
| 5’→3’ polymerase activity | T4DNAPolymerase, Klenow Fragment , | 5’-overhangs |
| 3’→5’ exonuclease activity | T4 DNA Polymerase, Klenow Fragment | 3’-overhangs |
| 3’-phosphatase activity | T4 PNK | 3’-P in sonicated DNA |
| 5’-kinase activity | T4 PNK | Non-phosphorylated DNA (e.g., PCR product) |
Figure 1 | Blunting and phosphorylation of different types of DNA ends
| Hazardous | No |
| Quality Control | Endo- and Exonucleases The End Repair Enzyme Mix storage buffer and 10X End Repair Reaction Mix were tested for the absence of contaminating endonucleases and exonucleases using the labeled oligonucleotide test. Functional testing Blunting efficiency: 3’- and 5’-overhanging oligoduplexes incubated with 2.5 µL of End Repair Enzyme Mix in 50 µL of 1X End Repair Reaction Mix for 5 min and 20 min at 20°C in the presence of [γ-33P]-ATP, then separated on denaturing PAGE and detected by phosphoimaging, resulted in ≥ 95% of expected bands specific for the blunt end reaction product. Phosphorylation efficiency: incubation of 5 µg of 200 bp dephosphorylated DNA fragment with 2.5 µL of End Repair Enzyme Mix in 50 µL of 1X End Repair Reaction Mix for 5 min and 20 min at 20°C, followed by column purification, subsequent ligation and analysis on gel resulted in ≥ 98% of higher molecular weight bands compared to non-ligated DNA. |
| Storage Condition | -20 C |
Obsah balení
50 rxns
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