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

488 Preston
1. 10× PCR buffer: 100 mM Tris-HCl, pH 8.3; at 25°C, 500 mM KCl, 15 mM MgCl 2 ; and
0.1% w/v gelatin. Incubate at 50°C to melt the gelatin, filter sterilize, and store at –20°C
(see Note 2).
2. dNTP stock solution (1.25 mM each of dATP, dGTP, dCTP, and dTTP) made by diluting
commercially available deoxynucleotides with PCR water.
3. Thermostable DNA polymerase, such as Amplitaq DNA Polymerase (Perkin–Elmer Cetus,
Norwalk, CT) supplied at 5 U/µL.
4. Mineral oil.
5. A programmable thermal cycler machine, available from a number of manufacturers,
including Perkin–Elmer Cetus, MJ Research, and Stratagene.
6. Degenerate oligonucleotide primers should be purified by reverse-phase high-performance
liquid chromatography or elution from acrylamide gels, dried down, resuspended at
20 pmol/µL in PCR-water, and stored at –20°C, preferably in aliquots.
7. The DNA template can be almost any DNA sample, including a single-stranded cDNA
from a reverse transcription reaction, DNA from a phage library, and genomic DNA. The
DNA is heat denatured at 99°C for 10 min and stored at 4 or –20°C.
8. Chloroform (see Note 3).
9. Tris-saturated phenol (see Note 3), prepared using ultra-pure redistilled crystalline phenol
as recommended by the supplier (Gibco-BRL [product #5509], Gaithersburg, MD). Use
polypropylene or glass tubes for preparation and storage.
10. PC9 (see Note 3): Mix equal volumes of buffer-saturated phenol, pH >7.2, and chloroform,
extract twice with an equal volume of 100 mM Tris-HCl, pH 9.0, separate phases by
centrifugation at room temperature for 5 min at 2000g, and store at 4 to –20°C for up
to 1 mo.
11. AmAc (7.5 M) for precipitation of DNA. Ammonium acetate is preferred over sodium
acetate because nucleotides and primers generally do not precipitate with it. Dissolve in
water, filter through 0.2-µm membrane, and store at room temperature.
12. 100% ethanol stored at –20°C.
13. 70% ethanol stored at –20°C.
14. TE: 10 mM Tris, 0.2 mM EDTA, pH 8.0. Dissolve in water, filter through 0.2-µm
membrane, and store at room temperature.
15. 50× TAE: 242 g of Tris-HCl base, 57.1 mL of acetic acid, 18.6 g of Na 2 (H 2 O) 2 EDTA.
Dissolve in water, adjust volume to 1 L, and filter through 0.2-µm membrane. Store at
room temperature.
16. HaeIII-digested φX174 DNA markers. Other DNA molecular weight markers can be used
depending on availability and the size of the expected PCR-amplified products.
17. 6× gel loading buffer (GLOB): 0.25% bromophenol blue, 0.25% xylene cyanol FF, 1 mM
EDTA, and 30% glycerol in water. Store up to 4 mo at 4°C.
18. Agarose gel electrophoresis apparatus and electrophoresis grade agarose. For the optimal
resolution of DNA products >500 bp in length, NuSieve GTG agarose (FMC BioProducts)
is recommended.
19. Ethidium bromide (EtBr; see Note 3). Ethidium bromide (10 mg/mL stock) prepared in
water and stored at 4°C in a brown or foil-wrapped bottle. Use at 0.5 to 2.0 µg/mL in water
for staining nucleic acids in agarose or acrylamide gels.
20. For the elution of specific PCR-amplified DNA products from agarose gels, several methods
are available, including electroelution and electrophoresis onto DEAE-cellulose membranes
(12,13). Several commercially available kits will also accomplish this task. I have had
some success with GeneClean II (Bio 101, La Jolla, CA) for PCR products >500 bp
in length, and with QIAEX (Qiagen, Chatsworth, CA) for products from 50 to 5000 bp. If
you do not know the approximate size of the PCR-amplified products and wish to clone