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

478 Horton, Raju, and Conti-Fine
the proper reading frame with the His tag. It ends with “ta”; the “a” added by the
polymerase finishes the termination codon (taa) and is included in the HindIII site.
2. Materials
1. cDNA template: This was reverse transcribed from total RNA using Superscript RNase
H-reverse transcriptase (Life Sciences) and an oligo (dT) primer using manufacturers
instructions.
2. Reagents for PCR: 10× buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3; red sucrose is
a PCR-compatible gel-loading dye consisting of ~1 mM cresol red in 60% sucrose (6);
Taq DNA polymerase, 10 mM dNTP mix, 10 mM MgCl 2 solution (Perkin–Elmer Cetus,
Norwalk, CT).
3. Reagents for agarose gel electrophoresis.
4. GeneClean (Bio 101, La Jolla, CA).
5. Ligation reagents: T4 DNA ligase (Stratagene, La Jolla, CA) 8 U/µL, ligase buffer supplied
by manufacturer, 10 mM ATP (Pharmacia, Piscataway, NJ), and PEG 8000 (Aldrich,
Milwaukee, WI). Recently, we have used T4 ligase buffer from Life Technologies (Gibco-
BRL, Gaithersburg, MD), which already contains ATP and PEG.
6. T-linker oligonucleotides:
a. Nde-T-linkers: TL-A: (23-mer) 5′-agcttgaattcgcggccgcatat-3′; TL-B: (18-mer)*
5′-tatgcggccgcgaattca-3′;
b. His-T-linkers: HisTL-A: (55-mer) 5′-gcggccgcatatgggatcctcacatcatcatcaccatcactcgagtggccaagct-3′;
HisTL-B: (21-mer) *5′-gcttggccactcgagtgatgg-3′
The 5′ end of each “B” oligonucleotide is phosphorylated (represented by the *) so
that it can be ligated to the (nonphosphorylated) end of the PCR product. The 5′ end
of the “A” oligonucleotide is not phosphorylated. The “B” oligo only needs to be long
enough to bind the “A” oligo during the ligation and in the annealing steps of the early
rounds of reamplification. A 5′ overhang on oligo “A” is not a problem, because this is
filled in by the polymerase during reamplification.
c. Dissolve primers at a stock concentration of 10 µM, which is generally considered
20× for PCR. After mixing T-linker primers, they are at a final concentration of approx
5 µM for ligation reactions.
7. Ampligrease (see ref. 7): plain petroleum jelly, Vaseline brand, or generic is suitable.
Apply quality control checks as described (7).
8. Reagents for bacterial culture, including competent Escherichia coli, appropriate antibiotic,
LB agar.
9. 95% ethanol containing 2% (w/v) potassium acetate.
10. 75% ethanol.
3. Methods
3.1. PCR
The HisT-linker was used to clone the coding region of the α3 subunit of the
nicotinic acetylcholine receptor from human bronchial epithelium (manuscript in
preparation; see Note 2) into the E. coli expression vector pT7-7 (8). The following
sequence-specific primers were used:
Vb8-1cpe5:
hpVbe3:
ha3cpe5:
ha3cpe3:
5′-ggagttatccagtcacc-3′
5′-gggaattcgtcgactgctggcrcagarrta-3′
5′-ccagtggccagggcctcaga-3′
5′-tatgcatcttccctggccatca-3′