| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| This protocol employs the activity of T4 DNA polymerase to blunt 3' overhangs on DNA fragments. This protocol contains directions for both small- and large-scale applications (1 to 10, and 10 to 30 μg of DNA, respectively). T4 polymerase can also be used to fill in 5' overhangs. T4 DNA polymerase catalyzes both 5' to 3' DNA synthesis activity and 3' to 5' exonuclease activity. The exonuclease activity is more active than that of DNA polymerase I (unlike DNA polymerase I from E. coli, T4 DNA polymerase does not have a 5' to 3' exonuclease activity). |
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1. Restriction enzyme digest the DNA (see protocol for Restriction Enzyme Digesting DNA) and add an equal volume of Phenol (CAUTION! see Hint #1).
2. Mix well by inversion, centrifuge for 2 min in a microcentrifuge to separate the phases and save the aqueous phase (top layer).
3. To the aqueous phase, add an equal volume of Chloroform:Isoamyl Alcohol.
4. Mix well by inversion, centrifuge in a microcentrifuge to separate the phases and save the aqueous phase (top layer).
5. To the aqueous phase, add 2 volumes of 100% Ethanol and incubate at -70°C for 30 min (see Hint #1).
6. Centrifuge for 15 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:
Small Reaction (1 to 10 μg DNA)
5 μl of 10X T4 DNA Polymerase Buffer
2.5 μl of 2mM dNTPs
2.5 μl of BSA Solution
2 to 20 μl of DNA (1 to 10 μg of DNA)
5 Units of T4 DNA Polymerase
20 to 38 ddH2O (final reaction volume is 50 μl)
Incubate at 37°C for 15 min.
Inactivate at 65°C for 5 min.
Large Reaction (10 to 30 μg)
10 μl of 10X T4 DNA polymerase buffer
5 μl of 2mM dNTPs
5 μl of BSA Solution
20 to 60 μl of DNA (10 to 30 μg of DNA)
10 Units of T4 DNA Polymerase
20 to 60 ddH2O (final reaction volume is 100 μl)
Incubate at 37°C for 15 min.
Inactivate at 65°C for 5 min.
9. Add an equal volume of 100% Phenol (see 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. To the aqueous phase, add an equal volume of Chloroform:Isoamyl Alcohol.
12. Mix well by inversion, centrifuge in a microcentrifuge to separate the phases and save the aqueous phase (top layer).
13. Either do a) OR b) below:
a). Clean up the aqueous solution by using a BioGel P-10 and save the eluate (follow the manufacturer's instructions on the use of a BioGel P-10 column).
To the eluate, add one-tenth volume of 3 M Sodium Acetate and 3 volumes of 100% Ethanol (continue with Step #14).
--OR--
b) Add one-fifth volume of 10 M of 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 the tubes on paper towels and allow sample to air dry.
17. Resuspend the DNA in a small volume of TE Buffer and run DNA on an agarose gel (see protocol on DNA Gel Electrophoresis).
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| dNTPs |
| Prepared in ddH2O
2 mM dCTP
2 mM dGTP
2 mM dATP
2 mM dTTP
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| BSA Solution |
| in ddH2O
2 mg/ml Bovine Serum Albumin
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| T4 DNA Polymerase Buffer (10X) |
| 5 mM DTT
0.1 M Magnesium Acetate
0.66 M Potassium Acetate
0.33 M Tris-Acetate, pH 7.9
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| Chloroform:Isoamyl Alcohol |
| (CAUTION! see Hint #1)
24:1 Chloroform:Isoamyl Alcohol
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| TE Buffer |
| 10 mM Tris
pH 8.0
1 mM EDTA
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Ethanol
Bovine Serum Albumin (BSA)
Magnesium Acetate
Sodium Acetate
Restriction Enzyme
Potassium Acetate
Tris
DNA Polymerase, T4
Isoamyl Alcohol
Chloroform
Phenol
dCTP
dTTP
dGTP
Ammonium Acetate
dATP
DTT
EDTA
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. 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.
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