John M. S. Bartlett.pdf - Bio-Nica.info

Cloning PCR Products 389
3.4.2. Dephosphorylation of Vector DNA
1. To 1 µg linearized M13 DNA in 10 µL TE, add CIP 0.05 U for 5′ overhangs, 0.5 U for
3′ overhangs or blunt ends, 5 µL of 10× CIP buffer, and H 2 O to 50 µL of total volume.
2. Incubate at 37°C for 60 min.
3. Inactivate CIP by heating the reaction to 75°C for 10 min in the presence of 5 mM EDTA,
pH 8.0.
4. Extract the reaction once with phenol:chloroform and recover DNA by sodium acetate/
ethanol precipitation as in Subheading 3.2., steps 4–6. Dissolve the DNA pellet in
20 µL of TE.,
5. Check recovery of M13 vector and insert DNA by electrophoresing an aliquot of each
through 0.8% agarose.
3.5. Ligation of PCR Products into M13 Vectors (see Notes 10 and 11)
1. Set up the ligation reaction and add components in the following order: 50 ng of M13
vector DNA, 1 µL of 10 mM ATP (1 mM final), 1 µL of 10× ligation buffer, 1– 4 µL of
DNA insert (3- to 5-fold molar excess), 5 U T4 DNA ligase for blunt termini, 1 U T4 DNA
ligase for cohesive termini, and H 2 O to 10 µL of total volume.
2. Set up a negative control ligation in which an equal volume of water is substituted for
the PCR DNA insert and a positive control ligation containing an appropriate blunt- or
cohesive-ended restriction fragment, preferably of a similar size to the PCR product.
3. Incubate overnight at 14°C for cohesive-end ligations or at room temperature for blunt-end
ligations.
4. Transform 2.5 to 5 µL of the ligation reaction into E. coli-competent cells and plate in a
soft agar overlay containing 0.33 mM IPTG and 0.03% X-gal. Identify recombinant phage
clones by blue/white selection (see Note 11).
4. Notes
1. The use of a spin filtration unit to purify the PCR product from unincorporated nucleotides
and primers is only recommended if the PCR mixture does not contain unwanted species
larger than the nucleotide cutoff value of the unit. For the Microcon-100, this corresponds
to 300 bases (single-stranded) or 125 bp (double-stranded). If larger unwanted products are
present, the target product should be purified by electrophoresis through low-melting-point
agarose followed by adsorption to glass beads.
2. If a spin filtration device is not available, PCR products can be partially purified from
residual primers and dNTPs by precipitation with sodium acetate/ethanol, followed by
a 70% ethanol wash. Better removal of such reactants can be achieved by adjusting to
2 M ammonium acetate and adding 2 vol of ethanol, although it should be noted that
ammonium ions are a strong inhibitor of T4 DNA polymerase and must be thoroughly
removed by extensive washing in 70% ethanol before the end-repair step.
3. Occasionally, PCR products end-repaired and kinased as described may fail to clone as
blunt-ended molecules, most probably because of the persistence of Taq DNA polymerase
bound at DNA termini. In this situation, residual enzyme can be removed by adding to
the sample 50 µg/mL proteinase K in 10 mM Tris-HCl, pH 7.8, 5 mM EDTA, 0.5% (v/v)
SDS, and incubating at 37°C for 30 min. Extract with phenol/chloroform, and precipitate
PCR products with sodium acetate/ethanol.
4. It is important not to exceed the recommended amount of T4 DNA polymerase enzyme or
the incubation time of 20 to 30 min for the end-repair reaction, since both may result in
excessive exonuclease activity and nonblunt “nibbled ends.” T4 DNA polymerase also has
excessive exonuclease activity at higher temperatures (37°C).