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

Cloning Gene Family Members 495
2. Set up the ligation reactions with the vector and insert similar to the following:
a. Reaction 1: 1 µL of vector (10 ng; vector control);
b. Reaction 2: 1 µL of vector + 1 µL of insert (~10 ng insert);
c. Reaction 3: 1 µL of vector + 4 µL of insert.
Then add 2 µL of 5× T4 DNA ligase buffer (Gibco-BRL) and water to 9.5 µL. If the
buffer is more than 4-mo-old, the ATP may be depleted. Therefore, add fresh ATP to a
final concentration of 1 mM.
3. For cohesive-end ligations add 0.5 µL of T4 DNA ligase (1 U/µL), gently mix, spin 5 s
in a microfuge, and incubate at 15°C for 10 to 20 h. For blunt-end ligations add 1 µL
of T4 DNA ligase (5 U/µL), gently mix, spin 5 s in a microfuge, and incubate at 25°C
(or room temperature) for 1 to 12 h. Stop the reaction by heating at 75°C for 10 min and
store the samples at –20°C.
4. Set up a bacterial transformation with competent DH5α bacteria or a comparable strain
of bacteria. Be sure to include a positive control (10 ng of undigested vector DNA) and a
negative control (water). To 1.5-mL microfuge tubes, add half of the ligation mix (5 µL)
or 5 µL of control DNA or water and 50 µL of competent bacteria (thawed slowly on ice);
incubate on ice for 30 min. Heat-shock at 42°C for 2 min. Return to ice for 1 min. Add
200 µL of LB media containing 10% glycerol. Mix gently and allow bacteria to recover
and express the ampicillin resistance gene by incubating at 37°C for 1 h.
5. Prewarm LB-Amp plates at 37°C for 45 min. About 30 min before plating the bacteria on
the plates, add 40 µL of X-Gal and 4 µL of IPTG and quickly spread over the entire surface
of the plate using a sterile glass spreader. Spread 20 to 200 µL of the transformation
reactions on these plates. Allow the inoculum to absorb into the agar and incubate the
plates inverted at 37°C for 12 to 24 h (see Note 4). Afterward, placing the plates at 4°C
for 2 to 4 h will help enhance the blue color development.
3.3.4. Plasmid DNA Minipreps and DNA Sequencing
1. Colonies that contain active β-galactosidase will appear blue, whereas those containing
a disrupted LacZ gene will be white. Set up minicultures by inoculating individual white
colonies into 2 mL of LB media containing ampicillin. After growing at 37°C overnight,
isolate the plasmid DNA. Resuspend the DNA in 20 to 50 µL of water or TE.
2. Digest 5 to 20 µL of the DNA with the appropriate restriction enzymes and analyze by
agarose gel electrophoresis (see Subheading 3.2.3.).
3. Perform double-stranded DNA sequencing on recombinants containing inserts in the
expected size range.
4. Notes
1. All PCRs should be set up in sterile laminar flow hoods using pipet tips containing filters
(aerosol-resistant tips) to prevent the contamination of samples, primers, nucleotides,
and reaction buffers by DNA. If the PCR is going to be reamplified by PCR, all possible
intervening steps should also be performed in a sterile hood with the same precautions to
prevent DNA contamination. These precautions should also be extended to all extractions
and reactions on the nucleic acid (RNA or DNA) through the last PCR. Likewise, all
primers, nucleotides, and reaction buffers for PCR should be made up and aliquoted using
similar precautions. All buffers for PCR should be made with great care using sterile
disposable plastic or baked glass, and restricted for use with aerosol-resistant pipet tips.
2. Standard PCR buffers contain 15 mM MgCl 2 (1.5 mM final concentration). In many
cases, changes in the MgCl 2 concentration will have significant consequences on the
amplification of specific bands. In PCR-amplifying the four exons of the AQP1 gene,