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388 Walsh
PCR products are now flush-ended and phosphorylated at their 5′ ends, ready for
direct cloning into SmaI-cut, dephosphorylated M13 vector or for concatamerization,
as required.
3.3. Concatamerization/Digestion of PCR Products (see Notes 5–8)
1. To 10 µL PCR product, add 2 µL of 10× T4 DNA ligase buffer, 7 µL 60% PEG 8000 (20%
final), and 2 U T4 DNA ligase. Incubate at room temperature overnight.
2. Increase the volume to 100 µL with water, and perform one extraction with
phenol:chloroform. Avoiding the white PEG precipitate at the interface, remove 10 µL
of the aqueous phase to check extent of concatamerization by electrophoresis through
0.8% agarose. Run out alongside an aliquot of the original PCR product and DNA size
markers (see Note 8).
3. If concatamerization is judged to be successful (PCR product present as trimers and
larger species), extract the remaining 90 µL once with chloroform/isoamyl alcohol and
precipitate with sodium acetate/ethanol as above. Dissolve in 20 µL TE.
4. Add 10 U of appropriate restriction enzyme(s), 3 µL 10× reaction buffer, and H 2 O to
30 µL. Incubate at the required temperature for 1 h.
If cutting with two enzymes that have different salt requirements, digest with the
low-salt enzyme first, heat-inactivate at 65°C for 20 min (or phenol-extract heat-stable
enzymes), then adjust salt concentration with 1 M NaCl, and add 10 U of the second
enzyme. Where two enzymes require completely different buffers, phenol/chloroformextract
and sodium acetate/ethanol-precipitate the DNA in between each digest.
5. Check to ensure the digest now contains only monomer-size PCR product by agarose gel
electrophoresis. If larger species remain, indicating incomplete digestion of concatamers,
add more enzyme and continue digestion.
6. When digestion is complete, electrophorese the digest mixture through 0.8% low-meltingpoint
agarose and recover the PCR product by glass bead isolation using Geneclean.
3.4. Preparation of M13 Vector DNA for Ligation
3.4.1. Digestion with Restriction Endonucleases
Where digestion of the vector with two enzymes is required, the ability of each
enzyme to cleave toward the end of linear DNA molecules should be considered to
determine the preferred order of sequential addition New England Biolabs catalog,
Reference Appendix; (see Note 9). Enzymes that cut less efficiently toward DNA
termini should be used first. In this situation where directional cloning is required,
digest M13 derivatives containing the polylinker in both orientations, for example,
M13mp18 and M13mp19.
1. Set up the following digest: 1 µg M13 RF DNA, 5 U restriction enzyme, 2 µL 10× reaction
buffer, and H 2 O to 20 µL.
2. Incubate for 1 h at the appropriate temperature (25°C for SmaI and 37°C for all other polylinker
enzymes). Remove 1 µL to analyze extent of digestion by electrophoresis through
0.8% agarose. If digestion is not complete, add more enzyme and continue incubation.
3. When complete, extract the digest once with phenol:chloroform and precipitate DNA with
sodium acetate/ethanol as in Subheading 3.2., steps 4–6. Dissolve the DNA pellet in 10 µL
of TE and digest with a second enzyme if required.
M13 DNA cut with a single enzyme should now be treated with calf intestinal
alkaline phosphatase to reduce recircularization during ligation.