1. Prepare PCR solution (2000ml)

• 200ml 10x Taq polymerase buffer (with MgCl₂)
• 160ml dNTP (2.5 mM) --- 200mM final concentration
• 200ml 5'-primer (10mM) --- 1mM final concentration
• 200ml 3'-primer (10mM) --- 1mM final concentration
• 1240ml double distilled water

2. Place yeast cells (roughly equal to a grain of sand) from colonies in 12 sets of 8-PCR tube strips (Midwest Scientific).

3. Place lids on tube strips and microwave the samples for 1 minute 30 seconds (Wach, A., Brachat A., Pohlmann, R. Phillipsen, P. 1994, Yeast 10 1793-1808). Immediately place them on ice. It is easier to use a 96-well tray (cat# 403081, PE Applied Biosystems) already on ice, then place the tube strips on it.

4. Add 10ml of Taq polymerase (cat# M2665, Promega Corp.) to the prepared PCR solution (0.5 ml /100ml solution), and mix well.

5. Using a multichannel pipetter (Finnpipette), add 20ml of PCR solution into each tube. Votex the tubes in tray for 20 seconds and check that solutions are turbid. Mixed solutions should be on ice until they are placed in a thermal cycler (the reaction should begin within 15 minutes or less after step 3).

6. Place the 96-well tray with PCR mixture in a pre-heated thermal cycler and start PCR.

The program of this reaction is:

Step	Temperature (°C)	Time (min.)
1	94.0	2:00
2	92.0	0:30
3	50.0	0:30
4	72.0	3:00
5	Repeat steps 2-4 29 times
6	72.0	10:00
7	4.0	0:00

7. Centrifuge the samples at 4,000rpm for 2 minutes (Centrifuge 5810 R, Eppendorf).

8. Using a multichannel pipetter, add 7ml of sample, while taking care not to disturb the pellet, to 4ml dye (25mg bromophenol blue, 25mg xylene cyanol, 7ml double distilled water, and 3ml glycerin). Mix well by pipetting up and down.

9. Run gel electrophoresis to detect the specific DNA fragment.