PROCEDURE FOR TESTING AND ASSAYING SNAP MARKERS[Testing of SNAP primer sets] [Assaying SNAP markers]
Testing of SNAP Primer Sets
We determined experimentally that primers that show specificity in two sets of PCR reactions that differ by 10 PCR cycles are specific over a 1,000-fold range of template DNA concentration. Therefore, primer specificity is tested by running two identical sets of PCR reactions per primer pair, in which the PCR reactions differ only in the number of cycles used during the amplification process. Primer pairs displaying presence of a PCR product on agarose gels for the specific allele and the absence of a PCR product for the non-specific allele in both sets of PCR reactions are suitable for use as molecular markers. For further details see Drenkard et al. (2000). Plant Physiology 124: 1483-1492.
Materials and Reagents
Materials and Reagents
Plant DNA micro-prep
- Extraction buffer: 200 mM Tris-Cl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS.
- TE: 10 mM Tris, 1.0 mM EDTA pH 8.0
- 70% ethanol
- Vortex mixer
- Polypropylene pestles (Kontes pellet pestle, available from Fisher Scientific)
- 1.5 ml microcentrifuge tubes
- Genomic DNA samples
- 2.5 mM dNTPs: 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP
- Forward and reverse primers (60 ng/µl each) for both alleles
- AmpliTaq Gold DNA polymerase (Perkin Elmer, Branchburg, NJ) - AmpliTaq Gold DNA polymerase 10X buffer
- Thermal cycler
- Tubes or 96-well plates that fit the thermal cycler
Micro-Preparation of Plant Genomic DNA
(Modified by Charles Gasser's lab from Edwards et al.,1991. Nucleic Acids Research 19: 1349).
1. Place 400 µl of extraction buffer into a microcentrifuge tube and shut the cap of the tube on a leaf to clip out a section of tissue.
The amount of tissue used is approximately 0.007 grams. Rosette leaves are optimal but cauline leaves also work. Wear gloves and minimize handling.
2. Grind the tissue using disposable polypropylene pestles that fit exactly into 1.5 microcentrifuge tubes. Grind the tissue until is well mashed and a significant amount of chlorophyll has been released into the buffer.
The use of a drill-like variable speed motor allows processing of up to 100 samples in a day by one person. Pestles can be soaked in 0.5 M HCl, rinsed with water, autoclaved and re-used.
3. Spin 5 min. in a microcentrifuge at 14,000 rpm.
4. Transfer 300 µl of supernatant to a fresh tube avoiding plant tissue carry over, and precipitate nucleic acids by adding 300 µl of isopropanol. Vortex briefly.
5. Spin 5 min. in microcentrifuge at 14,000 rpm and discard supernatant. Rinse the DNA pellet with 1 ml of 70% ethanol.
6. Carefully remove the supernatant and briefly air-dry the final pellet. Resuspend it in 100 µl of TE buffer.
1. For each primer pair to be tested mix the following reagents in a microcentrifuge tube:
1.0 µl 2.5 mM dNTPs
1.0 µl 60 ng/µl forward primer
1.0 µl 60 ng/µl reverse primer
2.0 µl 10X AmpliTaq Gold DNA polymerase buffer
0.2 µl (1 unit) AmpliTaq Gold DNA polymerase
13.8 µl water
2. Dispense 18 µl aliquots into microcentrifuge tubes or 96-well plates to be used for amplification reaction. To each tube add 2µl of DNA samples prepared by the method described above.
3. Insert the tubes/plates into a thermal cycler, and amplify using the following cycle: 5 min. at 94oC, (30 sec. at 94oC, 1 min. at 62oC) 35 times, finally 10 min. at 72oC. If available, set ramp speeds at 2o/s to 94oC and 1.4o/s to 62oC.
When using SNAP markers for mapping, the number of cycles used for the PCR reaction is very important at guaranteeing the specificity of the primers. If primer pairs were tested using 28 and 38 cycles, reactions used for mapping should be run using 35 cycles.
4. Add 2 µl of DNA loading dye. Separate the products on a 1.5 % (w/v) agarose gel.