Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Filling 5' Extensions to Create Flush Termini for Cloning (Klenow Reaction) |
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| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley | ||||||||||||||||||||||||||||||||||||||
| Procedure |
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1. Take DNA after restriction enzyme digest and add an equal volume of Phenol. 2. Mix well by inversion centrifuge for 2 min in a microcentrifuge to separate the phases and save the aqueous phase (top layer). 3. Add an equal volume of Chloroform:Isoamyl Alcohol to the aqueous phase. 4. Mix well by inversion centrifuge for 2 min in a microcentrifuge to separate the phases and save the aqueous phase (top layer). 5. Add two volumes of 100% Ethanol to the aqueous phase and allow the DNA to precipitate by incubating at -70°C for 1 to 5 hours. 6. Centrifuge for 2 min in a microcentrifuge to pellet the DNA and discard the supernatant. 7. Resuspend DNA pellet in ddH2O to a final concentration of approximately 500 μg/ml. 8. Set up the following reaction (depending on the amount of DNA to be processed): Small Reaction (1 to 10 μg of DNA) 5 μl of 10X Nick Translation Buffer 2.5 μl of 2mM dNTPs 2.5 μl of BSA Solution 2 to 20 μl of DNA (1 to10 μg of DNA) 1-5 U Klenow Fragment 20 to 38 ddH2O (final reaction volume is 50 ul) Large Reaction (10 to 30 μg of DNA) 10 μl of 10X Nick Translation Buffer 5 μl of 2mM dNTPs 5 μl of BSA solution 20 to 60 μl of DNA (10 to 30 μg of DNA) 1-5 U Klenow Fragment 20 to 60 ddH2O (final reaction volume is 100 ul) Incubate at room temperature for 30 min 9. Add an equal volume of 100% Phenol (CAUTION! See Hint #1 and (Hint #2). 10. Mix well by inversion centrifuge for 2 min in a microcentrifuge to separate the phases and save the aqueous phase (top layer). 11. Add an equal volume of Chloroform:Isoamyl Alcohol to the aqueous phase. 12. Mix well by inversion centrifuge for 2 min in a microcentrifuge to separate the phases and save the aqueous phase (top layer). 13. Clean up the aqueous solution with a BioGel P-10 and save the eluate and perform either a) OR b) below: a) Add one-tenth volume of 3 M Sodium Acetate and 3 volumes of 100% Ethanol to the eluate (continue with Step #14). --OR-- b) Add one-fifth volume of 10 M Ammonium Acetate and 3 volumes of 100% Ethanol (continue with Step #14). 14. Incubate at -70°C for 30 min. 15. Centrifuge for 15 min in a microcentrifuge to pellet the DNA and decant the supernatant. 16. Invert tubes on paper towels and allow sample to air-dry. 17. Resuspend DNA in a small volume of TE Buffer and analyze the DNA on an agarose gel (see Protocol ID#1265). |
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| Solutions |
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| BioReagents and Chemicals |
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Sodium Acetate Bovine Serum Albumin (BSA) dCTP dTTP Magnesium Sulfate dGTP dATP Isoamyl Alcohol Tris Chloroform Ammonium Acetate Phenol EDTA DTT Ethanol |
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| Protocol Hints |
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1. You can increase the sample volume by adding ddH2O before the Phenol and Chloroform extractions if you find it difficult to extract in very small volumes. 2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. |
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