实验步骤

1. Protocol for grinding plant seeds.

   1) grind using a bead mill

      a. place one seed into each well of a 2 ml square well block.

      b. Pipette nuclease-free water into the well according to the following volumes:

         800 ul for soybean or similar sized seeds

         600 ul for cotton or similar sized seeds

         200 ul for canola, sorghum, wheat, or similar sized seeds

         100ul for arabidopsis or similar sized seeds

      c. Place a 4 mm stainless steel grinding ball in each well of the 2 ml 96 square well block and cover with sealing mat. Place block in the bead mill and shake at 1,500 rpm for 10 min.

      d. Continue to Extract seeds DNA.

   2) grind individually using a plastic pestle or mortar

      a. place one seed into a 1.5 ml microcentrifuge tube.

      b. Pipette nuclease-free water into the well according to the above step b.

      c. Incubate the seed with water for 1 hour at 55℃.

      d. Grind hydrated seeds in tube using a plastic pestle or mortar.

      e. Continue to Extract seeds DNA

   3) grind individually using liquid nitrogen

      a. grind seed into a fine powder in liquid nitrogen using a mortar and pestle.

      b. Transfer between 5 and 100 mg of ground seed material into a pre-weighed 1.5 ml microcentrifuge tube. Record the mass of the transferred seed material.

      c. Pipette 4 ul of nuclease-free water for every mg of transferred ground seed material into the sample tube and vortex to mix.

      d. Continue to Extract seeds DNA

   4) Extract of ground seeds DNA

      a. Add 45 μl PS1 Buffer and 5 μl PS2 Buffer to the collection tube. Close the tube and vortex briefly. Make sure the ground seeds is covered by the Extraction Solution.

      b. Incubate at 56°C for 10-20 minutes.

      c. Incubate at 95°C for 5 minutes.

      d. Add 50 μl PS3 buffer and vortex to mix.

      e. Store the extraction at 2-8°C.

2. Protocol for whole plant seeds.

   1) Add 45 ul – 95 ul PS1 Buffer and 5 μl PS2 Buffer to the collection tube. Close the tube and vortex briefly. Make sure the ground seeds is covered by the Extraction Solution.

   2) Incubate at 56°C for 1 hours.

   3) Incubate at 95°C for 5 minutes.

   4) Add 50 -100 μl PS3 Buffer and vortex to mix.

   5) Store the extraction at 2-8°C.

3. PCR Protocol

This protocol serves as a guideline for PCR amplification. Optimal reaction conditions, such as incubation times, temperatures, and amount of template DNA, may vary and must be individually determined.

   1) Thaw primer solutions. Keep on ice after complete thawing, and mix well before use.

   2) Mix the PCR Master Mix by vortexing briefly. It is important to mix the PCR Master Mix before use to avoid localized differences in salt concentration.

   3) Prepare one of the following reaction mixes on ice: (For a 25 μl reaction volume)

  4) Gently mix the reaction and spin down in microcentrifuge.

  5) Set up program for a routine PCR reactions:

      Initial Denaturation 94-95°C for 1-5 min

      25-40 cycles            94-95°C for 30 sec

                                        45-70°C for 10-30 sec

                                        72°C for X min(1min/kb)

      Final extension 72°C for 5 min

      Final soak 4-10°C

   6) For a simplified hot start, proceed as described in step 7. Otherwise, place the PCR tubes in the thermal cycler and start the cycling program.

   7) Simplified hot start: Start the PCR program. Once the thermal cycler has reached 94°C, place the PCR tubes in the thermal cycler. In many cases, this simplified hot start improves the specificity of the PCR.