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

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3.3. Cloning and DNA Sequencing of PCR-Amplified Products
3.3.1. Preparation of Vector for Ligation
1. For blunt-end ligations, digest 1 µg of pBluescript II KS phagemid DNA (Stratagene) with
10 U EcoRV in a 50-µL vol. Incubate at 37°C for 2 h. For cohesive-end ligations, similarly
digest the vector with the appropriate restriction enzyme(s).
2. For both blunt-end ligations and cohesive-end ligations where the vector has been digested
with only one restriction enzyme, it is necessary to remove the 5′-phosphate from the
vector to inhibit the vector from self ligating. This is accomplished by treating the vector
with CIP according to the manufacturer’s recommendations. Note that 1 µg of a 3-kbp
linear DNA molecule contains 1 pmol of 5′ overhangs (BamHI), blunt-ends (EcoRV), or
3′-overhangs (PstI), depending on the enzyme that digested it. Afterward, add EDTA to
5 mM and heat-kill the enzyme at 65°C for 1 h. Adjust the volume to 50 to 100 µL with TE
and extract once with Tris-saturated phenol, twice with PC9, and twice with chloroform.
Back extract each organic layer with 50 µL of TE and pool with the final sample.
AmAc-EtOH precipitate (see Subheading 3.2.2.) and resuspend in 10 µL of water.
3. If the insert is going to be directionally cloned into the vector, just extract once with
50 µL of PC9, AmAc-EtOH precipitate (see Subheading 3.2.2.) and resuspend in 10 µL
of water.
3.3.2. Preparation of Inserts for Ligation
AmpliTaq and other thermostable DNA polymerases often fail to completely fill in
the ends of the double-stranded DNA products, thus leaving recessed 3′ termini that
can be filled in with the Klenow fragment of E. coli DNA polymerase I. This should be
done whether or not the DNA is going to be digested with restriction enzymes added to
the ends of the primers for directional cloning (see Subheading 3.1.).
1. AmAc-EtOH precipitate the DNA (see Subheading 3.2.2.) and resuspend in 15 µL of
water.
2. Add 2 µL of 10× restriction enzyme reaction buffer. Klenow DNA polymerase works
well in most restriction enzyme digestion buffers (10× REact 2 or 3 from Gibco-BRL).
If the DNA is going to be subsequently digested with a restriction enzyme(s), use the
buffer for that enzyme.
3. Add 2 µL of 10 mM dNTP solution. Then, add Klenow DNA polymerase (1 U/µg DNA)
and incubate at room temperature for 15 min.
4. Heat-inactivate the enzyme at 75°C for 10 min. If the DNA is going to be directly used in
ligation reactions, it is not necessary to purify the DNA from the unincorporated dNTPs
because they will not inhibit T4 DNA ligase. To concentrate the DNA sample, proceed
with step 6.
5. PCR products containing restriction sites on their ends should now be digested with the
restriction enzymes. Incubate in the appropriate buffer, using 20 U of enzyme/µg of DNA
and incubating for 2 to 4 h at the proper temperature.
6. Extract the DNA once or twice with PC9 and precipitate with AmAc-EtOH as described
above (see Subheading 3.2.2.). Resuspend the final pellet in 5 to 10 µL of water.
3.3.3. DNA Ligation and Bacterial Transformation
1. At this point, it is often advantageous to run a small aliquot of the different DNA fragments
on a gel to assess their approximate concentrations and purity. Ideally, you want at least a
21 molar ratio of insert to vector in the ligation reactions. If necessary return to the above
procedures to isolate more DNA for the ligation reaction.