DNA extraction protocol

Materials

• Promega DNA extraction kit
• 1.5 ml Microcentrifuge Tubes
• 2.0 ml Microcentrifuge tube
• Steel Beads (3.2 mm Dia, 90g. S.S.Beads. Catalog # 11079132ss. www.biospec.com)
• Water Bath, 65o C
• Water Bath, 37oC
• Isopropanol, room temperature
• 70% Ethanol, room temperature

This protocol is modified based on protocol of the Wizard Genomic DNA Purification Kit from Promega.

Procedure

1. Put on gloves, and label two screw cap microtubes (2 ml) with the sample names, your team ID, and your initials “S#T#Initial”. put the microtubes on the plastic tube rack
2. Use forceps to add 4 steel beads into each 2 ml microtube
3. Select one or more healthy-looking Arabidopsis leaf and use a scissor to cut the leaves off the plant
   • Note:
     • One leaf for the WT plant
     • 3-4 leaves for the PLIP3-OX plant
4. Chop the leaves briefly with scissors and put them into the corresponding pre-labeled 2 ml microtubes
5. Using a P1000 micropipette, add 600 μl of Nuclei Lysis Solution into the tube with leaves (tissues)
6. Vortex the tube on the vortexer flat attachment for 10-15 minutes to grind the tissue
7. Incubate the sample at 65oC for 15 minutes
8. Using a P20 micropipette, add 3 μl of RNase Solution to the tube and mix it by inverting it 2-5 times
9. Incubate this mixture at 37oC for 10 minutes. Then let the sample cool for 5 minutes before proceeding
10. Add 200 μl of Protein Precipitation Solution, and vortex vigorously at max speed for 20 seconds
11. Centrifuge the sample for 3 minutes at 13,000 x g
    • The precipitated proteins will form a tight pellet at the bottom of the tube
    • Please handle the samples gently, and don’t disturb the pellet
12. Using a micropipette carefully remove the supernatant containing the DNA and transfer it to a clean 1.5 ml microtube, make sure to label the tube with your team ID
    • Do not touch the Protein Pellet at the bottom
    • The tube with beads can be discarded in solid waste
13. Add 600 μl of room temperature isopropanol to the new tube with supernatant inside
14. Gently mix the solution by inversion until thread-like strands of DNA form a visible mass
    • You may not see a visible mass forming, continue with the experiment
15. Centrifuge the sample at 13,000 x g for 2 minutes at room temperature
16. Dump the liquid into the waste beaker, and keep the tube that has the DNA at the bottom (might be invisible)
17. Add 600 μl of room temperature 70% ethanol to the microtube from step 16, then gently invert the tube several times to wash the DNA. Centrifuge the sample at 13,000 x g for 2 minutes at room temperature
18. Gently pour the ethanol out into the liquid waste beaker
    • Be careful, don’t dump the DNA pellet out
19. Invert the tube onto a clean absorbent paper and air-dry the pellet for 15 minutes
20. Add 25 μl of DNA Rehydration Solution to rehydrate the DNA overnight at 4°C
    • Please put your samples into the storage box labeled “BS171 Sxx” on the front demonstration bench
    • We will help you to put the samples at 4°C and then freeze them for the next lab