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

RAPD Fingerprinting 119
14. GeneAmp ® Thin-Walled Reaction Tubes (0.6 mL), designed for optimal fit in the DNA
thermal cycler or DNA cycler 480 sample block.
15. Agarose (Sigma or similar product).
16. Ethidium bromide to visualize DNA
17. TBE buffer: 1 M Tris, 0.9 M boric acid, 0.01 M EDTA, pH 8.3.
18. Loading dye (6× concentration: 0.25% bromophenol blue, 0.25% xylene cyanol FF, and
40% (w/v) sucrose in water, keep at 4°C.
19. Electrophoretic size standards: PCR marker, 50–2000 bp, 123-bp DNA ladder, Supercoiled
DNA ladder [2–16 kb], Sigma.
3. Methods
Programming of the thermal cycler for RAPD analysis is described first, followed
by the PCR conditions used for the reaction.
3.1. RAPD PCR Programming
Program the thermal cycler (PerkinElmer model 480, which is a thermocycler with an
average ramp speed) to denature for 1 min at 94°C, anneal for 2 min at 27°C (see Note 1),
and allow primer extension for 2 min at 72°C for the first five cycles. Then, program
for 45 cycles of 1 min denaturation at 94°C, 2 min of annealing at 32°C (see Note
1), and 2 min primer extension at 72°C. Set the final extension period for 15 min
at 72°C. With a thermocycler with a faster ramp speed, like the PerkinElmer model
9600, a shorter protocol can be used, such as 45 cycles of 15 s at 94°C, 30 s at 27°C,
and 1 min at 72°C.
3.2. Setting Up the PCR for RAPD Analysis
The following protocol is particularly suitable for RAPD analysis of C. albicans.
Minor modifications of this method are useful for RAPD analysis of other Candida species.
The components and procedure of the RAPD reaction mixture are listed as follows:
1. Thaw the PCR buffer, MgCl 2 solution, dNTPs, and primer solutions on ice and mix
properly before use.
2. Prepare the PCR master mix in GeneAmp Thin-Walled Reaction Tubes containing
approximately 50 ng of C. albicans genomic DNA (see Note 6), 5 µL of 10× PCR standard
buffer, 200 µM dNTPs, 5 µL of 25 mM MgCl 2 (see Note 3), 1 µM of primer, 1.5 U Taq
polymerase (Sigma, see Note 4), and make up to 50 µL of final reaction volume with
double distilled deionized sterile water.
3. Mix these reagents well by vortexing and then overlay with mineral oil (40 µL) to prevent
evaporation and internal condensation. Spin for a short period before amplification until a
smooth interface appears between the aqueous and the mineral oil layer.
4. Preparation of the agarose gel is as follows: Separate the amplified products in 2% agarose
gel because the molecular weight of the resultant amplicons are in the range of 0.3 to 4.2 kb
(agarose has to be dissolved in the same electrophoresis running buffer, generally, 1× TBE.
Include 0.5 µg/mL ethidium bromide during the preparation of the gel).
5. Sample preparation for gel loading is as follows: Retrieve amplified products by adding
150 µL of chloroform and pipetting out the aqueous droplet or by placing the reaction
mixture over a piece of parafilm. Then, mix well the retrieved aqueous amplification
mixture (12.5 µL) with the loading buffer (1.5 µL) and load into wells in an agarose
gel together with an appropriate DNA size marker (ranging in size from 50 to 6 Kb) in
a standard manner.