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Fast DNA End Repair Kit


This kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.
  
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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
Table 1 | Enzymatic activities of the End Repair Enzyme Mix
Activity Enzyme providing the activity Type of DNA ends repaired
5’→3’ polymerase activityT4 DNA Polymerase, 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

Figure 1 | Blunting and phosphorylation of different types of DNA ends

  
Storage Condition-20 C
HazardousNo
Quality ControlEndo- 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.
Blunting and phosphorylation of different types of DNA ends

Blunting and Phosphorylation

Blunting and phosphorylation of different types of DNA ends

Figure 1 | Blunting and phosphorylation of different types of DNA ends


End repair of different types of DNA ends

End repair of different types of DNA ends

End repair of different types of DNA ends

Figure 2 | End repair of different types of DNA ends Oligoduplexes with different types of DNA ends (3’-, 5’-overhangs and blunt non-phosphorylated ends) were repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier’s recommendations in the presence of [γ-33P]-ATP. Reaction products were separated on denaturing polyacrylamide gel and phosphoimaged.

Note: the two oligos of the oligoduplex migrate as two separate bands under a denaturating conditions. Non-specific end repair reaction products appear as additional bands of unexpected length.
C1, C2 – synthetic radiolabeled oligoduplex identical to expected reaction product
S1 – synthetic radiolabeled oligoduplexes identical to reaction substrate with 3’-overhangs
S2 – synthetic radiolabeled oligoduplexes identical to reaction substrate with 5’-overhangs
1 – oligoduplex end repaired with Fast DNA End Repair Kit (20°C, 5 min)
2 – oligoduplex end repaired with Fast DNA End Repair Kit (20°C, 20 min)
A – oligoduplex end repaired with Vendor A (20°C, 30 min)
B – oligoduplex end repaired with Vendor B (RT, 45 min; 70°C, 10 min)


Efficient ligation after DNA end repair

Efficient ligation after DNA end repair

Efficient ligation after DNA end repair

Figure 3 | Efficient ligation after DNA end repair with the Fast DNA End Repair Kit A 130 bp PCR product with 3’- dA overhangs was end-repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier’s recommendations and subsequently ligated with T4 DNA Ligase (#EL0014). Ligation conditions: 200 ng of 130 bp PCR product ligated with 5U T4 DNA Ligase in 1X T4 DNA Ligase Buffer with 5% PEG 4000 for 1 h at 22°C.
M – GeneRuler 1 kb Plus DNA Ladder (#SM1333)
C1 – PCR product
C2 – PCR product after ligation
T – PCR product after end repair using Fast DNA End Repair Kit (20°C, 5 min) and ligation
A – PCR product after end repair using Vendor A kit (20°C, 30 min) and ligation
B – PCR product after end repair using Vendor B kit (RT, 45 min; 70°C, 10 min) and ligation.


Protocols and Product Manuals

pdf   Fast DNA End Repair Kit - Product Information