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

Cloning Gene Family Members 489
all of them, the QIAquick-spin PCR purification kit is recommended (Qiagen) because
this will remove all nucleotides and primers before attempting to clone. This kit is
also recommended for purification of PCR products for secondary PCR-amplification
reactions.
2.3. Cloning and DNA Sequencing of PCR-Amplified Products
1. From Subheading 2.2., items 8–14 and 20.
2. pBluescript II phagemid vector (Stratagene). A number of comparable bacterial expression
vectors are available from several companies.
3. Restriction enzymes: EcoRV (for blunt-end ligation).
4. Calf intestinal alkaline phosphatase (CIP; New England Biolabs, Beverly, MA).
5. Klenow fragment of Escherichia coli DNA polymerase I (sequencing grade preferred) and
10 mM dNTP solution (dilute PCR or sequencing grade dNTPs).
6. T4 DNA ligase (1 or 5 U/µL) and 5× T4 DNA ligase buffer (Gibco-BRL).
7. Competent DH5α bacteria. Can be prepared (12,13) or purchased. Other bacterial strains
can be substituted.
8. Ampicillin: 50 mg/mL stock in water, 0.2-µ filtered, and stored in aliquots at –20°C
(see Note 4).
9. LB media: 10 g of bacto-tryptone, 5 g of bacto-yeast extract, and 10 g of NaCl dissolved in
1 L of water. Adjust pH to 7.0. Sterilize by autoclaving for 20 min on liquid cycle.
10. LB-Amp plates: Add 15 g of bacto-agar to 1000 mL of LB media before autoclaving for
20 min on the liquid cycle. Gently swirl the media on removing it from the autoclave to
distribute the melted agar. Be careful: The fluid may be superheated and may boil over
when swirled. Place the media in a 50°C water bath to cool. Add 1 mL of ampicillin,
swirl to distribute, and pour 25 to 35 mL/90-mm plate. Carefully flame the surface of the
media with a Bunsen burner to remove air bubbles before the agar hardens. Store inverted
overnight at room temperature, then wrapped at 4°C for up to 6 mo.
11. IPTG: Dissolve 1 g of isopropylthiogalactoside in 4 mL of water, filter through 0.2-µm
membrane, and store in aliquots at –20°C.
12. X-Gal: Dissolve 100 mg of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside in 5 mL of
dimethylformamide and stored at –20°C in a foil wrapped tube (light sensitive).
13. Plasmid DNA isolation equipment and supplies (12,13) or plasmid DNA isolation kits,
available from many manufacturers.
14. Double-stranded DNA sequencing equipment and supplies (12,13) or access to a DNA
sequencing core facility.
3. Methods
3.1. Design of Degenerate Oligonucleotide Primers
1. The first step in designing a degenerate primer is to select a conserved amino acid sequence
and then determine the potential nucleotide sequence (or the complement of this sequence
for a downstream primer), considering all possible permutations. If the amino acid
sequence is relatively long, you can potentially design two or more degenerate primers.
If only one is made, make it to sequences with a high (50–65%) GC content because
these primers can be annealed under more stringent conditions (e.g., higher temperatures).
Figure 1 shows an alignment of the amino acid sequences for several members of the
Aquaporin gene family in the two most highly conserved regions. Also shown is the
consensus amino acid sequence, the degenerate nucleotide sequence, and the sequence
of the primers we used to isolate Aquaporin gene family members. Interestingly, not
only are these two regions highly conserved among the different members of this gene