Protocol DNA

DNA isolation

Method A Qiagen

B CTAB

1. L. brownii Hangju A 22 2. L. brownii Hangju A 160 3. L. brownii Hangju A 161 4. L. brownii Hangju A 165 5. L. brownii Hangju A 167 6. L. brownii Hangju B 31 7. L. brownii Hangju B 87 8. L. brownii Hangju B 253 9. L. brownii Hangju B 270 10. L. brownii Hangju B 271 Under the clean bench:

1. Prepare petri-dishes with Nr. and write the No. on the cap. 2. sterilized work: cut leaves tissues, put them into petri-dishes. 3. DNA isolation(10 samples) A-Procedure:

1. Disrupt the sample material (≦100 mg wet weight or ≧20 mg lyophilized tissue ) using the TissueRuptor, the TissueLyser, or a mortar and pestle.

2. Add 400μl Buffer AP1 and 4μl RNase A. Vortex and incubate for 10 min at 65℃. Invert tube 2-3 times during incubation. Note: Do not mix Buffer AP1 and RNase A before use. 3. Add 130μl Buffer AP2. Mix and incubate for 5 min on ice.

Recommended: Centrifuge the lysate for 5 min at 20000*g (14000 rpm) 4. Pipet the lysate into a QIAshredder Mini spin (pink color) column in a 2ml collection tube. Centrifuge for 2min at 20000*g (14000rpm) 5. Transfer the flow-through fraction into a new tube without disturbing the pellet. Add 1.5 volumes of Buffer AP3/E, and mix by pipetting. 6. Transfer 650μl of the mixture into a DNeasy Mini spin (white color) column in a 2 ml collection tube. Centrifuge for 1 min at≧6000*g(≧8000 rpm). Discard flow-through. Repeat this step with the remaining sample.

7. Place the spin column into a new 2 ml collection tube. Add 500μl Buffer AW, and centrifuge for 1 min at≧6000*g. Discard flow-through. 8. Add another 500μl Buffer AW. Centrifuge for 2 min at 20000*g. Note: Remove the spin column from the collection tube carefully so the column does not come into contact with the flow-through.

9. Transfer the spin column to a new 1.5ml or 2ml microcentrifuge tube, and add 100μl Buffer AE for elution. Incubate for 5 min at room temperature. Centrifuge for 1 min at ≧6000*g. Repeat this step.

B-Procedure:

1. I. The case of DNA isolation with liquid N2: a. Switch on incubator or water bath at 60℃. b. Prepare tube with label. c. weight 1.0g of plant material (just leaves). d. Prepare extraction buffer (put 2% ß-Mercaptoethanol into extraction buffer (2*CTAB) before use under the hood: ex.2ml/100ml(v/v)).e.Put 5ml of extraction buffer into 15ml Falcon tube. f. Set the working place (liquid N2, 100ml beaker, chemical spoon.g. bring frozen Mortar and Pestle from -80℃ freezer. h. pour liquid N2 into frozen mortar and grain the plant material fine with frozen pestle. i .freeze chemical spoon with liquid N2)

II. DNA isolation of dried plant material: a. Switch on incubator or water bath. b. Prepare tube(Label), c. weight 400mg of plant material (ca.40% of fresh material weight (1.0g)=dried material weight (ca.400mg)). d. Prepare of extraction buffer (Put 2% ß-Mercaptoethanol into extraction buffer before use: 2ml/100ml(v/v)).

2. Put 1.0g/fresh (400ml/dried) of powder into 15ml Falcon tube containing 5ml extraction buffer containing ß-Mercaptoethanol) and mix it by vortex.

3. Incubate the tube in 65℃ water bath for 1 hour.

4. Prepare to label the genotype on 15ml new falcon tube and 2ml eppendorf tube.

5. Put 1ml of 100% ethanol into 2ml eppendorf tube.

6. After incubation let it to be cool.

7. Add 5ml of Chloroform&isoamylalcohol(49:1)mixture into tube under hood.

8. Shake it strongly.

9. Centrifuge it at RT with 3000g(6000 rpm) for 15 min. 10.Transfer the supernatant (5ml) into new tube. 11.Add 80% volume of supernatant of isopropanol(4ml). 12.Gently it mix.(Be careful! Gently)

13.Hook DNA clumps with J-form Pasteur-Pipette from solution and then put it into a 2ml eppendorf tube containing 1ml of 100% ethanol.# Best quality DNA can be received from hooking step.

14.(not essential) The pellet in ethanol solution could be stored at -20℃ until use.

15.Discard ethanol(or centrifuge the mixture when the pellet is not formed).

16.Wahing the pellet with 1ml of 76% Ethanol containing 0.2M sodium cetate (washing solution I) by inverting the tube. 17.Discard the washing solution I.

18.Washing the pellet with 1ml of 76% Ethanol containing 10mM Ammonium acetate.

19.Discard the washing solution II.

20.Dry the pellet in clean bench at room temperature (RT). Alternatively

the rest ethanol could be removed by pipette and then the pellet could be dried at 60℃ for 5 to 10 min. Completely dried pellet is not good to be dissolved again.

21.Dissolve the DNA pellet with di-sterilized water or TE buffer(10mM Tris-HCl pH8, 1mM EDTA) containing 1μl of RNase (10mg/ml) by incubating at 60℃ (ThermoMixer) with 1400 rpm for 15 min. C. Measure DNA concentration

Prepare: 200μl tube, Pipette, Tips, Centrifuge, Vortex, Schott bottle(500ml), magnetic bar, gelcastle(100ml), 12-comb (6x), Nitrile gloves, Scanner, Uv-lamp, Electrophoresis apparatus, Power supply di-sterilized water, loading dye (Orange G), 10mg/ml of Ethidium bromide, Agarose, 0.5xTBE buffer. Be careful! Ethidium bromide is toxic!

1. Prepare the 0.8% agarose gel: weight 0.8g agarose powder (per 100ml of 0.5xTBE buffer) and close the lid half (not tightly) and cook this mixture in microwave for 3 min. The solution is transparent when the agarose completely be melted. The agarose solution should be cooled until 50℃ on the stirrer. During cooling set the gel castle. With regular distances, put 6 times of 12-comb into gel castle. Add 10μl of ethidium bromide into agarose solution (100ml) and stir it completely. Pour the agarose solution into gel castle and let it until polymerization(ca.20 min). 2. Sample preparation: First dilute original DNA 10x (ex. 10μl+90μl) and

then put 3μl of dilute DNA into 200μl tube and add 12μl of loading dye mixture (1.5μl of 10x loading dye Orange G：10.5μl of di-sterilized water ). Vortex the tube shortly. Spine it down.

3. Electrophoresis: Take out the polymerized gel from castle. Put the gel into buffer tank of electrophoresis apparatus containing 0.5x TBE buffer. Load 10μl out of 15μl of DNA loading dye mixture into gel well(with comparing standard maker). Close the lid and connect the cable into power supply(DNA(-charged) is running from – (black) to+(red) ). Check the polar and run the power supply (150V) for 150 min. Turn off the power supply and check DNA under the UV-lamp. Scan the gel and save the gel picture(.gel). DNA concentration adjustment in the agarose gel. Final concentration: 500ng/μl.