I. Description

II. Protocols

A. Blunting Reaction
B. Dephosphorylation of Vector DNA
C. Ligation Reaction

I. Description

This kit allows the conversion of DNA fragments with 3'- and 5'-protruding ends to DNA fragments with blunt ends. Conversion of the two types of protruding ends can be accomplished simultaneously by the 3'->5' exonuclease and 5'->3' polymerase activities of T4 DNA Polymerase. The resulting blunt-ended DNA can be ligated efficiently into a ovector using the included DNA Ligation Kit Solutions. The reactions can be used directly for bacterial transformation and in vitro packaging procedures without further purification.

1. The DNA Blunting Kit can fill dephosphorylated 5'-protruding ends, but not
the phosphorylated 3'-recessed ends that are often present in sonicated DNA designed for use in shotgun cloning experiments.
2. Ligation Solutions A and B should be thawed on ice and mixed thoroughly immediately before use.
3. It is not necessary to extract the blunted, ligated DNA with phenol prior to bacterial transformation and other reactions. DNA can be ethanol precipitated to concentrate the sample.

II. Protocols

A. Blunting Reaction

1. Combine the following in a microcentrifuge tube:

^a DNA should have been purified by ethanol precipitation.

2. Incubate at 70°C for 5 minutes.
3. Add 1 µl of T4 DNA Polymerase and mix gently by pipetting.
Do not vortex.
4. Incubate at 37°C for 5 minutes. If the DNA has a low G+C content, incubate at 25°C instead of 37°C.
5. Add DNA Dilution Buffer to bring the DNA concentration to 1 µg/50 µl and mix thoroughly. If the DNA concentration is already low, proceed directly without diluting the DNA. Place on ice to ensure deactivation of T4 DNA Polymerase. If the solution is to be stored, immediately extract DNA with phenol/chloroform and precipitate with ethanol. The precipitate should be dissolved in DNA Dilution Buffer and stored at -20°C.

B. Dephosphorylation of Vector DNA

Fragments generated by restriction digestion have a 5'-phosphate even after they have been treated with T4 DNA Polymerase for blunting. Since the insertion of DNA fragments into vector DNA proceeds most efficiently when the 5'-ends of the vector DNA are dephosphorylated (minimizes self-circularization), vector DNA should be treated with an enzyme such as alkaline phosphatase. However, when PCR products are to be inserted, do not dephosphorylate the vector unless phosphorylated primers were used for PCR. See Section II.C. Insertion of DNA Fragments Into Plasmid Vector for details.

1. Prepare > 10 µg of blunt-ended vector DNA (see Section II.A). Precipitate the vector DNA with ethanol.
2. Combine the following in a microcentrifuge tube:

^a If calf intestine phosphatase (CIAP) is preferred, use 2 µl of CIAP (10-20 U/µl) in place of bacterial alkaline phosphatase (BAP).

3. Incubate at 65°C for 30 minutes when using BAP. Incubate at 50°C for 30 minutes if using CIAP.
4. Add 150 µl of phenol/chloroform (1:1) and mix well.
5. Centrifuge. Transfer the upper layer to a fresh tube.
6. Repeat steps 4 and 5.
7. Add 7.5 µl of 3 M NaCl (final concentration 150 mM).
8. Add 375 µl (2.5 volumes) of cold ethanol. Leave at -20°C for 30-60 minutes.
9. Centrifuge and discard supernatant.
10. Rinse precipitate with 1 ml of cold 70% ethanol. Dry under reduced pressure.
11. Dissolve precipitate in TE buffer. If the DNA is to be used immediately for ligation, resuspend in DNA Dilution Buffer.

C. Ligation Reaction

Insertion of DNA Fragments Into Plasmid Vector

1. Prepare 5-10 µl of a DNA solution containing the blunt-ended DNA fragment and dephosphorylated vector DNA dissolved in DNA Dilution Buffer. The DNA fragment:vector DNA molar ratio should be 3-10:1.

There are several points to consider when PCR products are being inserted into vector DNA. First, the blunting reaction should be performed on the PCR products before ligation because PCR products will have single 3' dA-nucleotide overhangs [Clark (1988) Nucleic Acids Res. 16: 9677]. Second, since the 5'-ends of PCR products usually originate from non-phosphorylated synthetic primers, the vector DNA must provide the 5'-phosphates and therefore should not be dephosphorylated. Third, the recommended molar ratio of PCR product:vector is >10:1.

2. Add 4-8 volumes (20-80 µl) of Ligation Solution A and mix well.
3. Add 1 volume (5-10 µl) of Ligation Solution B and mix well.
4. Incubate at 16°C for 30 minutes.
5. Proceed directly to bacterial transformation using up to 20 µl of the above solution for 100 µl of competent cells. If transformation by electroporation is desired, precipitate the DNA with ethanol and resuspend in an appropriate buffered salt solution. Do NOT use the ligation solution directly for electroporation, as it will generate sparks with the use of pulsed electric fields.

Self-Circularization of Linear Blunt-Ended DNA

Proceed essentially as described above, except use lower concentrations of DNA to ensure high efficiency of intramolecular ligation. Keep the volume of the ligation mixture as small as possible to obtain high transformation yields.

Insertion of Linker DNA into Blunt-Ended Vector DNA

Proceed essentially as described above. If the linker has a high A+T content or is shorter than 8 bases, carry out the ligation reaction at 4-10°C for 1-2 hours.

Insertion of DNA into Blunt-Ended l DNA

1. Combine dephosphorylated l vector DNA^a and the DNA to be inserted in a total volume of 5-10 µl. Supplement with NaCl to give a final concentration of 300 mM NaCl. The salt concentration is important because it promotes a high yield of concatemeric l DNA, which is favorable for in vitro packaging.

2. Add 1 volume (5-10 µl) of Ligation Solution B to the DNA solution and mix
well.

3. Incubate at 26°C^b for 5-10 minutes.^c

4. The ligation reaction mixture (up to 5 ml) can be used directly in l in vitro
packaging reactions.^d

Notes:
^a In general, 1-2 µg of l phage vector DNA is used, but similar results can be obtained with as little as 250 ng.
^b Ligations involving l phage vector DNA work better when carried out at 26°C than at 16°C.
^c Ligation reactions set up as above are usually complete after 5-10 minutes of incubation. Longer reactions will not increase ligation efficiency.
^d Components of the ligation mixture will not inhibit l in vitro packaging reactions as long as < 5 µl are used with a standard packaging extract of > 25 µl. The DNA should be ethanol precipitated to reduce volume if more of the ligation mixture is to be packaged in a single reaction.