1. A Quick Overview of Lemaux Lab’s DNA Extraction Protocol Eric Trieu March 2009

2. What is DNA Extraction? Isolation of pure DNA Separation of DNA from lipids, proteins, sugars, organelles, RNA Especially important to remove DNAse, enzyme-inhibiting proteins, and inhibitory polysaccharides

3. How most people see DNA

4. How most people don’t see DNA Each phosphate carries a negative charge. Why?

5. A Few Caveats There are many different DNA extraction protocols Use depends on plant and source of tissue Also depends on intended downstream use (PCR, digestion, etc.) Because I don’t know much about how different protocols compare with each other, I will talk only about our protocol.

6. Leaves For DNA extraction, best to use young leaves from healthy tissue Younger leaf tissue in sorghum is near proximal end of leaf, near the stalk –Why? Younger leaf tissue has less polysaccharide & phenols

7. DNA extraction from older tissue DNA extraction from younger tissue

8. For each plant you want to temporarily store to extract in near future, either excise entire leaf and store in a bag, or obtain just enough leaf tissue to store in a 1.5 ml Eppendorf tube. All plant tissue should be stored in -80ºC freezer. Tissue can also be lyophilized. Label everything!!! THIS IS SUPER IMPORTANT!!! Long-term storage of leaf tissue

9. Grinding Leaves To grind leaves, either use mortar & pestle (manual grinding), or automated grinding (Qiagen shaker). Sonicators can also be used to lyse cells SAMPLES SHOULD BE KEPT FROZEN AT ALL TIMES, MEANING TUBE SHOULD BE REDIPPED INTO LIQUID NITROGEN EVERY 2-3 MINS TO KEEP DEGRADATIVE ENZYME ACTIVITY AT MINIMUM

10. QIAGEN shaker 1.Place leaf tissue in 1.5 to 2 ml Eppendorf tube unless tissue is already in the tube. Use roughly this much tissue in 1.5 mL centrifuge tube.

11. QIAGEN Shaker 2.Make sure samples are frozen. 3.Place samples inside “shaker tray” 4.Using metal tongs, dip tray containing samples into liquid nitrogen (WEAR SAFETY GLASSES) 5.Take tray out and add 2 metal balls to each tube. (When tubes are frozen, open as gently as possible) 6.Dip tray into liquid nitrogen again to keep tubes frozen (to make them easier to grind) 7.Place tray in grinder; set frequency to ~24-25 and timer to 45-60 seconds. If machine does not turn on, turn it off and back on. It should start after that. 8.Once grinding is complete, take tray out, pound it on table top to get all residue to fall to bottom. Dip tray into liquid nitrogen again.

12. Shaker Will samples at “A” verticle position or “C” position get ground more thoroughly? AC

13. ` These 8 samples came from Column.C on the shaker – less torque

14. Reagents 1.Extraction Buffer 2.SDS-detergent to solubilize membranes 3.Heating 4.Potassium acetate 5.1:1 Phenol:Chloroform 6.Isopropanol 7.TE + ribonuclease A ALWAYS mix thoroughly!

15. DNA Extraction Samples were ground on machine, tray pounded on bench and dipped into liquid N 2. Take tray out of liquid nitrogen; quickly remove cap to ensure tray and cap don’t stick together Tray removed from liquid N 2 Towel sheet used in case metal balls break cap.

16. DNA Extraction: Buffer + SDS Take samples out 2 at a time; add extraction buffer + SDS fast and vortex samples

17. Lipids in cell membrane SDS CTAB is one way to precipitate DNA. CTAB dissociates chromosomal DNA from binding proteins, and selectively precipitates nucleic acids. supposed to selectively remove polyphenolics and polysaccharides, which should be good for sorghum. SDS+CTAB are commonly used together; it forms micelles, like SDS.

18. Natural Extraction Buffer Natural extraction buffer contains NaCl, Tris-EDTA, H 2 0 What does each reagent do? Add 0.5–1% (v/v) of β-ME to extraction buffer immediately before use to decrease possibility of oxidation.

19. DNA Extraction Use magnets to pull out magnetic balls Repeat for the rest of samples Refreeze often Put samples in rack and put in 65 º C oven; check oven temperature to make sure it is at 65 Wait 15 mins; heat will allow SDS to solubilize membrane Immediately add KOAc to each tube, close tube and put on ice to allow SDS to precipitate.

20. Why NaOAc?

21. DNA Extraction At this point, you need to two more sets of tubes for transfer (e.g., if you have 10 samples, you will need 30 tubes, 20 you have not yet used) Centrifuge and transfer supernatant Do next step under hood! Phenol:Chloroform can be corrosive/harmful! Add phenol chloroform to each tube; shake vigorously Phenol Chloroform acts to remove proteins and certain other biomolecules but not DNA Certain methods use several extractions (e.g. two rounds of phenol chloroform, or one round of phenol chloroform and one round of chloroform to remove phenol NOTE: OXIDIZED PHENOL IS PINK/YELLOW. IF YOU SEE PINK/YELLOW IN THE PHENOL:CHLOROFORM SOLUTION, DO NOT USE IT

22. DNA Extraction: Supernatant After adding phenol chloroform and shaking vigorously, centrifuge. An aqueous layer will be on top, an organic layer on bottom, and a layer in the middle. Aqueous layer contains DNA and RNA. If accidentally get any of other layers, recentrifuge because proteins you extracted could degrade DNA Aqueous Layer Organic Layer Interphase Layer

23. DNA Extraction: Ispropanol After adding isopropanol, incubate at -20º C for at least 20 mins to allow DNA to precipitate, then centrifuge. A pellet will be at bottom each tube. What does isopropanol do? What else is in the solution that alllows isopropanol to do its job? Isopropanol, coupled with the neutralization effect of the potassium ions on DNA, will help to precipitate DNA by decreasing the dielectric constant of the solution. Generally, 1 equivalent of isopropanol is added to solution. Isopropanol is prefereed over EtOH, because more EtOH is needed to precipitate equivalent amounts of DNA.

24. DNA Extraction: Ethanol Remove isopropanol, add EtOH to wash, pipette out as much ethanol as possible Evaporate remaining EtOH (Use vacufuge to do this). Why is it a problem if ethanol is remaining? Add TER (TE+RNAse) and incubate at 37 º to let RNaseA degrade RNA. Store at -20 º C How long does purified DNA last in freezer?

25. Here’s where it worked…

26. Here’s where it didn’t work…What things could have gone wrong?

27. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide G C Allen, M A Flores-Vergara, S Krasynanski, S Kumar & W F Thompson