Protocol for Genotyping by PCR

PCR Setup:

(Per reaction)

Run on the GENE program in the MAMY folder on the thermal cycler.

This takes approximately 6 hours.

Digest DNA:

Set a water bath to 37 degrees.

Without changing reaction tubes, add the following directly to DNA from the PCR reaction. Place tubes in floating rack or float-boat for 2 hours.

To assay for an Box 3 (Sph I) mutant:

DNA from PCR	50
10x NE Buffer #2	6
Sph I Enzyme	3
dWater	1

To assay for an L985 (Hind III) mutant:

DNA from PCR	50
10x NE Buffer #2	6
Hind III Enzyme	3
dWater	1

To assay for a Triple (Avr II) mutant:

DNA from PCR	50
10x NE Buffer #2	6
Avr II Enzyme	3
100x BSA	0.6
dWater	0.4

Analizing the data (see diagram).

Enzyme	Size	Genotype
Avr II	463, 980	Triple Mutant
	1443	Wild-Type
Sph I	280, 453, 710	Box 3 Mutant
	453, 990	Wild-Type
Hind III	704, 739	L985 Mutant
	1443	Wild-Type