Resources Molecular Markers CAPS CAPS Protocol

PROCEDURE FOR ASSAYING CAPS MARKERS

[Materials and Reagents] [Experimental Protocols]

Materials and Reagents
Plant DNA mini-prep

- Extraction buffer: 100 mM Tris, 50 mM EDTA, 500 mM NaCl, 10 mM b-mercaptoethanol, pH 8.0
- 3.0 M sodium acetate adjusted to pH 5.2 with acetic acid
- BTE: 50 mM Tris, 1.0 mM EDTA pH 8.0
- TE: 10 mM Tris, 1.0 mM EDTA pH 8.0
- RNaseA: 10 mg/ml DNase free RNaseA dissolved in water
- 20% SDS
- 5.0 M potassium acetate
- Isopropanol
- Ethanol
- Liquid nitrogen
- Microcentrifuge
- Vortex mixer
- Water bath or incubator set at 65-70oC
- Ice
- Mortar and pestle
- 1.5 ml microcentrifuge tubes

CAPS reaction
- 2.5 mM dNTPs: 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP
- Forward and reverse primers
- Taq polymerase
- Taq polymerase 10X buffer
- Thermal cycler
- Tubes that fit the thermal cycler

Restriction enzyme digestions
- Restriction enzyme
- 10X concentrated restriction enzyme buffer
- Water bath or incubator set at the appropriate temperature for the restriction enzyme used


Experimental Protocols
Mini-Preparation of Plant DNA
1. Harvest approximately 0.1 g of fresh plant tissue and freeze in a 1.5 ml microcentrifuge tube using liquid nitrogen or dry ice.
The amount of tissue may vary between 0.05 and 0.2 grams, may consist of a mixture of leaves, stems, flowers or seedlings.
2. Grind the frozen tissue to a fine powder in a mortar and pestle.
3. Transfer frozen powder to a 1.5 ml microcentrifuge tube containing 0.5 ml of extraction buffer and vortex 10 seconds. Keep tubes on ice while processing the rest of the samples.
4 Add 35 µl of 20% SDS and vortex briefly. Incubate 10 minutes at 65-70oC.
5. Add 130 µl of 5.0 M potassium acetate. Mix thoroughly by shaking tube back and forth and incubate on ice for 5 minutes.
6. Spin 5 min. in a microcentrifuge at 14,000 rpm to remove undigested debris.
7. Transfer the supernatant to a fresh tube (approx. 700 µl), and precipitate nucleic acids by adding 60 µl of 3 M sodium acetate and 640 µl isopropanol.
From this step on the samples should be handled gently to decrease shearing of the DNA.
8. Spin 5 min. in microcentrifuge at 14,000 rpm and discard supernatant. Resuspend pellet in 200 µl BTE and spin again at 14,000 rpm to discard insoluble material.
9. Transfer supernatant to a fresh tube and reprecipitate by adding 1/10 vol. of 3 M sodium acetate and 2 vol. ethanol.
10. Resuspend pellet in 100 µl of TE and add 0.01 µg/µl RNaseA. Incubate 1 hour at 37oC or O/N at 4oC.
11. Reprecipitate DNA as in step 9 and centrifuge 10 min. at 14,000 in microcentrifuge. Wash the DNA with 70% ethanol and spin 5 min.
This second ethanol precipitation improves the amplification of the DNA in PCR reactions.
12. Carefully remove the supernatant and briefly air-dry the final pellet. Resuspend it in 50 µl of TE buffer.

CAPS reaction
1. For each DNA sample to be tested mix the following reagents in a microcentrifuge tube:
0.5 µl 2.5 mM dNTPs
1.0 µl 20 ng/µl forward primer
1.0 µl 20 ng/µl reverse primer
1.0 µl 10X Taq buffer
0.1 µl (0.5 units) Taq polymerase
5.5 µl water
2. Dispense 9 µl aliquots into microcentrifuge tubes to be used for amplification reaction. To each tube add 1µl of DNA samples prepared by methods described above.
The amount of DNA in 1 µl of a plant DNA mini-prep ranges from 10 ng to 100 ng, but the PCR reaction works well over this range. For cesium purified DNA samples 8 ng of DNA per reaction gives good results.
3. Insert the tubes into a thermal cycler, and amplify using the following cycle: 5 min. at 95oC, (1 min. at 95oC, 1 min. at 55oC, 2 min. at 72oC) 50 times, finally 10 min. at 72oC.

Restriction enzyme digestion and analysis of amplified products
1. For each sample to be tested, mix the following:
2 µl appropriate 10X restriction enzyme buffer
8 µl water
2-10 units appropriate restriction enzyme
2. Add 10 µl of the premix (from step 1) to each sample tube containing the amplified product and incubate at a suitable temperature for 2 hours.
3. Add 2 µl of DNA loading dye. Separate the products on an agarose gel.
Many of the CAPS markers can be successfully scored after electrophoresis in a standard 1.5% agarose gel. Resolution of fragments less than 500 bp long is greatly improved by using agarose gels consisting of 1% conventional agarose (for strength) and 2% NuSieve GTG agarose (supplied by American Bioanalytical, Natick, MA, USA). Other commercial agarose preparations that can be used at high concentrations should also give good results.