Authors
highQu
Summary
For an efficient blunt-end ligations, the nebulized, shared or restriction enzyme digested DNA has to be treated with blunting enzymes. Blunt-end PCR products after the high fidelity amplification have to be phosphorylated to enable the ligation reaction.
With a special blunting and phosphorylation enzyme blend, all kind of DNAs can be treated in one tube in a single procedure to achieve efficient blunting and phosphorylation prior to ligation. The procedure is simple and saves more than 60% of time in comparison to blunting and phosphorylation reactions performed separately.
Image 1 Introduction
HighEnd™ Repair Kit is a great tool for a rapid and highly efficient DNA end-repair before the ligation reactions. PCR products, shared or nebulized DNA, restriction-digested DNA and cDNA can be blunted/phosphorylated in a couple of minutes and are ready for an efficient blunt-end ligation and cloning.
The Kit includes HighEnd™ Repair Blend – an optimized mix of T4 DNA Polymerase and T4 Polynucleotide Kinase. The 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase form the blunt-ended DNA. T4 Polynucleotide Kinase phosphorylates 5′ DNA ends. The resulting DNA is a high quality blunt-ended substrate for T4 DNA Ligase.
Up to 1-5 microgram of the linear DNA can be blunted and phosphorylated in one 20 min reaction. After the thermal inactivation the reaction mixture can be used for blunt-end ligations.
HighEnd™ Repair Kit is an ideal choice for preparing for ligations the PCR products obtained with high fidelity polymerases.
Materials
1. HighEnd™ Repair Kit (highQu), includes following components:
a) HighEnd™ Repair Blend, 1 r/µl - an optimized mix of T4 DNA Polymerase and T4 Polynucleotide Kinase.
b) 10X HighEnd™ Buffer - contains contains Tris, NaCl, MgCl2, DTT, and other components.
c) 1 mM dNTP Mix
2. Test DNA solution (PCR products, shared or nebulized DNA, restriction-digested DNA, cDNA)
3. PCR-grade Water
Procedure
Important Notes
- Check the integrity and the concentration of the DNA prior the reaction.
- Always repurify PCR products before end-repair.
- Thaw and keep reagents on ice. Mix all components well before use.
- The optimal DNA amount in the reaction depends on the lengths of the DNA fragment. For example 1 µg of
- 1 kb linear DNA has ~3 pmol ends, but 1 µg of 100 bp linear DNA has 10 times more substrate for blunting/phosphorylation; i.e. even 30 pmol DNA ends. Therefore, the shorter is the DNA fragment, the less of it shall be used in micrograms for end-repair or for later ligation reaction.
- For high DNA amounts upscale the reaction accordingly. For example 5 µg of short 100 bp fragment can be end-repaired in 100 µl reaction using 2-5 µl of HighEnd™ Repair Blend.
Prepare a 25 µl end-repair reaction by adding following components:
a. Linear DNA in TE buffer or water - up to 1 µg (up to 30 pmol ends)
(1 µg of 1 kb linear DNA has ~3 pmol ends
1 µg of 0.5 kb linear DNA has ~6 pmol ends
1 µg of 0.1 kb linear DNA has ~30 pmol ends)
b. 10X HighEnd™ Buffer - 2.5 µl
c. 1 mM dNTP Mix - 2.5 µl
d. PCR Water - up to 24 µl
e. HighEnd™ Repair Blend, 1 r/µl - 1 µl (max 2 µl)
- Mix well; incubate for 20 - 30 min at 25°C.
- Inactivate enzymes at 75°C for 20 min and keep cooled in case the ligation is performed immediately or keep frozen in case the ligation is performed later.
- Alternatively, re-purify the DNA using PCR clean-up spin column kit, elute in 25 µl of water or TE and keep frozen.
- For subsequent ligation and cloning follow the recommendations of your ligation enzyme/kit.
Anticipated results
- DNA is ready for ligation in 20 minutes: PCR products, shared or nebulized DNA, restriction-digested DNA, cDNA.
- Conversion of all 5′- and/or 3′-protruding ends to 5′-phosphorylated blunt-ended ones
- Reproducible results
Further details
Download the HighEnd™ Repair Kit User Manual
References
http://www.highqu.com/products/enzymes/dna-related-enzymes/highend-repair-kit-167.html
Documents for download
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