(Adapted from Williams et al., 1990*)
NOTES: This protocol has been optimized for a specific set of conditions and the following precautions should be noted.
DNA isolations must include a phenol extraction (Option A, p.2).
Always resuspend DNAs in sterile ddH2O (Sigmaís Cell Culture Water, Cat. # W-3500, provides an excellent standard).
Accurate quantification of DNA amounts is important; fluorometric measurements are usually more accurate than spectrophotometric ones at low concentrations.
The concentration of all DNAs should be adjusted to 5 ng/μl, and they should be aliquoted in appropriate amounts and frozen until needed.
Take note of the lot number of the available Taq enzyme and perform the necessary experiments to determine the optimum amounts of enzyme and of MgCl2 for best performance. Repeat these tests whenever you change to a new lot of enzyme.
Use filtered pipette tips for the preparation of all stocks of reagents included in these protocols, as well as for pipetting DNAs and primers.
To ensure repeatability and consistent results, it is strongly recommended that all working solutions (primers, buffers, nucleotides) be prepared in advance for all planned experiments using the same reagents and high quality ddH2O. With this strategy, aliquots of the working stocks may be prepared for each experiment and stored frozen (-20•C); these would then be thawed only once, on the day of a particular experiment.
All conditions have been optimized for ERICOMP TwinBlockô system thermocyclers.
1. Prepare a bulk reaction mix containing all the components listed below except DNA and decamer primer.
2. Aliquot bulk mix into each labeled tube.
3. Add primer and DNA sample to each tube. Mix briefly (optional: centrifuge).
4. Overlay each sample with 2 drops or 50 μl of ultrapure mineral oil.
5. Place in PCR machine, making sure there is sufficient oil in each well to provide proper contact with tube.
6. Amplify using following program6 :
7. Optional: remove oil by adding 25 μl TE + 50 μl chloroform. Mix and centrifuge. Pipet top aqueous layer into new tube.
8. Add 5 μl 5X SGB to each tube. Mix well and centrifuge briefly.
9. Load 12 μl of each sample in a 2% agarose gel prepared with 1X TBE gel buffer (this gives resolution equal to novel agarose types).
10. Electrophorese at 35-40 mA, constant current, with buffer recirculation until the blue dye has migrated as required (approximately 3 h for 20 x 25 cm gels).
11. Stain gel in 1 μg/ml ethidium bromide (100 μl of 10 mg/ml ethidium bromide in 1000 ml dH2O) for 20 min with gentle shaking.
CAUTION: Ethidium bromide is extremely mutagenic ó wear double gloves when handling and use extra precaution.
12. Rinse gel in dH2O for 30 min, slide gel onto a UV transilluminator and photograph. For Fotodyne PCM-10 camera with 20 x 26 cm hood and Type 667 Polaroid film use an f8, 1†second exposure.
For Fotodyne MP-4 camera and Type 665 Polaroid (negative) film use an f8, 2†min exposure.
5X TBE Gel Buffer: 0.45 M Tris-borate, 10 mM EDTA
pH to 8.0 with glacial acetic acid or HCl.
A precipitate may form when stored for long periods of time.
Buffer: 0.9 M Tris-borate, 20 mM EDTA
pH to 8.0 with glacial acetic acid or HCl.
A precipitate may form when stored for long periods of time.
* Williams, J.G.K., A.R. Kubelik, D.L. Livak, J.A. Rafalski and S.V. Tingey. 1990. DNA polymorphisms amplified by rbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6536.
1 Sigmaís Cell Culture Water, Cat. # W-3500
2 It is essential to determine optimal concentrations of MgCl2 and Taq with each new lot of enzyme and DNA from pecies to be analysed.
3 Ultra pure.
4 Quantities may vary according to species source. Diluted in ddH2O (use Sigma water).
5 Ten-base pair primers can be obtained from OPERON Technologies, Inc.
6 Conditions optimized for ERICOMP TwinBlockô System thermocycler.
7 At 2400 m of altitude over sea level; You may increase slightly at lower altitudes.
