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

Cloning PCR Products 387
23. M13 RF DNA purchased from supplier or prepared in-house by CsCl density gradient
centrifugation.
24. Isopropyl-β-thiogalactopyranoside (IPTG): 20 mg/mL stock in sterile water, stored
at –20°C.
25. 5-Bromo-4-chloro-3-indolyl-βD-galactoside (X-gal): 20 mg/mL stock in dimethyl formamide,
stored at –20°C in glass vial.
26. Escherichia coli strain containing the F plasmid (e.g., JM101, JM107) and maintained
on M9 minimal agar.
3. Methods
3.1. Purification of PCR Products from Taq DNA Polymerase, Primers,
and dNTPs (see Notes 1 and 2)
1. Extract the completed PCR with an equal volume of chloroform. Spin in a microfuge at
13,000g for 2 min to separate the aqueous and organic phases.
2. Extract the upper aqueous phase twice with an equal volume of phenolchloroform and
once with an equal volume of chloroform/isoamyl alcohol.
3. Transfer the upper aqueous layer (up to 100 µL) into a Microcon unit housed in a 1.5-mL
Eppendorf tube and add 400 µL of TE buffer. Spin in a microfuge at 500g for 15 min
(Microcon-100) or at 14,000g for 6 min (Microcon-50). Add a further 400 µL of TE to
the sample reservoir, and spin as before. Volume retained will now be 50 to 100 µL. If
required (PCR product present in low yield), concentration down to a volume of 10 to 20
µL is achieved by a further spin cycle. Each cycle reduces the concentration of salts, PCR
primers, and dNTPs by approx 95%.
4. Invert the unit in a fresh tube and spin at 500g for 2 min to recover purified PCR product.
5. Check recovery by agarose gel electrophoresis of an aliquot of the concentrated product.
If amplified DNA is to be cloned by cohesive-end ligation via restriction sites
incorporated into PCR primers, and these sites are known to cut efficiently, the purified
product can now be digested with restriction endonucleases without further processing.
PCR products to be cloned by blunt-end ligation or via digestion with restriction
enzymes that cut inefficiently at DNA termini should be processed as follows.
3.2. Simultaneous End Repair and Phosphorylation
of PCR Products (see Notes 3 and 4)
1. Set up a reaction containing: 100 ng to 1 µg of purified PCR product, 3 µL of 10× T4 DNA
polymerase buffer, 100 µM each dNTP, 1 mM ATP, 0.5 U T4 DNA polymerase, 5 U T4
polynucleotide kinase, and H 2 O to 30 µL. Incubate at 25°C for 20 min.
2. Stop the reaction by incubating at 75°C for 10 min in the presence of 5 mM EDTA,
pH 8.0.
3. Increase the volume to 100 µL with H 2 O, and perform one extraction with phenol/
chloroform and one with chloroform/isoamyl alcohol.
4. Remove the aqueous phase to a fresh tube, and add 0.1 vol 3 M sodium acetate, pH 5.3, and
2.5 vol cold absolute ethanol. Mix well and store at –20°C for 1 h or at –70°C for 20 min.
Precipitate DNA by centrifugation at 13,000g for 10 min in a microfuge.
5. Remove the supernatant carefully and add 0.5 mL cold 70% ethanol to the pellet. Spin
again at 13,000g for 2 min. Discard the supernatant as before, vacuum dry the pellet
(2–5 min), and finally dissolve DNA in 10 µL of TE.