There are many good PCR kits available from different manufacturers. Check the instruction and use the buffers that accompany your kit.
Weizman Institute of Science BioGuide - PCR
10X PCR amplification Buffer (Sambrook, Fritsch and Maniatis, 1989)
500 mM KCl
100 mM Tris-HCl (pH 8.3 at room temperature)
15 mM MgCl2
0.1% gelatin
TAE Buffer (Tris-Acetate/EDTA Electrophoresis Buffer) (Sambrook, Fritsch and Maniatis, 1989)
1X Concentration:
0.04 M Tris-acetate
0.001 M EDTA
50X Concentrated stock (amount per liter) (Sambrook, Fritsch and Maniatis, 1989)
242 g Tris Base
57.1 mL of glacial acetic acid
100 mL of 0.5 M EDTA (pH8.0)
Download the RAPDistance program free from the web.
The program is a DOS based program and may not work on all computers. After trying it out we have come up with some additional hints for use. We have been able to downloaded it and used it through windows as well as starting the computer in DOS based mode. There are a number of .exe files and only some of them are needed for use in this exercise.
A number of help documents are included in the program. Open up help.txt, readme.doc or rapdtxt.doc for general help. Each of the batch programs includes a help document under Hbat.
Open the program by clicking on RAPDIN.exe or bat1. Follow the instruction to enter the data as listed in the exercise. Save the data with a name followed by .dat.
The next step is to make comparison between the data points to do this click on RAPDALG.exe or bat21.
Your most recent file is offered by default. You will be asked to save your file by a new extension name choose 2 so that your name will be saved with a new extension.
You will then be offered a variety of statistical programs. The manual recommends option 15. This generates 5 different files. To generate the tree you will need the .njt file.
To draw the tree click on 31bat. When it asks for the file name, give the name of your file with the .njt extension. This will be the basis for the production of the dendrogram in the file tdraw program. This saves the tree in an ASCI file. The file is labeled Tdraw.asc and can be opened in any word processing program. The Tdraw.asc file is overwritten with a new analysis, so save it under a new name if desired.