Plant Seed Direct PCR Kit
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实验步骤
1. Protocol for grinding plant seeds.
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
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.
d. Continue to Extract seeds DNA
4) Extract of ground seeds DNA
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.
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.
1) Thaw primer solutions. Keep on ice after complete thawing, and mix well before use.
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
