Isolation of Cassava Genomic DNA

Solutions

1. Extraction buffer (EB)

   	Stock	50 ml	100 ml	500 ml	1000 ml
   100 mM Tris-HCl pH 8.0 (ml)	1M	5	10	50	100
   500 mM NaCl (ml)	5M	5	10	50	100
   50 mM EDTA (ml)	0.5M	5	10	50	100
   SDS (1.25%)	20%	3.1	6.25	31.25	62.5
   dH20 to final volume		31.9	63.75	318.75	637.5

2. Autoclave for 15 min
3. Add 1% (w/v) of PVP, dissolve by mixing
4. Add 700 ul of b-mercaptoethanol to a liter of extraction buffer

Other reagents

• 20% SDS stock (w/v). Heat gently under stirring to accelerate dissolving. Autoclave and store at room temperature.
• 5M NaCl stock. Autoclave and store at room temperature
• 5M Potassium Acetate (ice-cold)
• 100 % Isopropanol (v/v). Store at –20°C
• RNase: 10 mg/ml

Procedure

1. Collect approximately 0.15 to 0.2g fresh young leaves in microfuge tubes. Add liquid Nitrogen and grind samples to a fine powder with a plastic pestle.
2. Add 850ml of EB at 65°C to each Eppendorf tube and mix gently with a plastic pestle till complete rehydration of the powder is achieved.
3. Shake vigorously until the tissue becomes dispersed in the buffer. Continue to mix for another 1 minute.
4. Incubate the Eppendorf tubes in a water bath at 65°C for 15 min and while mixing intermittently 5-6 times.
5. Remove tubes from water bath and allow it to cool to room temperature (~2 min.)
6. Add 250ml of ice-cold 5M potassium acetate and mix by gently inverting the tube 5-6 times then incubate on ice for 20 min.
7. Centrifuge at 12,000 rpm in eppendorf table-top centrifuge for 10 min
8. Transfer supernatant to a new 1.5 ml eppendorf tube
9. Add 1ml of 10mg/ml RNAse stock to each sample and incubate at 65% for 5 min
10. Add one volume of ice-cold Isopropanol (approx 700ml), and mix by gentle inversion 8-10 times
11. Incubate at -80°C for 1 hour and centrifuge at 12,000 rpm for 10 min.
12. Pour off supernatant and remove last drops of isopropanol by placing eppendorf facedown on paper towels
13. Resuspend pellet in 500ml of 50mM Tris-HCL/10mM EDTA (high salt TE).  (Hint:Incubating at 65°C is helpful for 10-15 min with constant gentle shaking to dissolve pellet)
14. Add one volume of ice-cold Isopropanol (500mL) and mix by gentle inversions 8-10 times
15. Incubate at -80°C for 1 hr and centrifuge at 12,000 rpm for 10 min
16. Pour off supernatant and remove last drops of isopropanol by placing face down on paper towels. Allow pellet to dry by leaving it on paper towel for another 1 hour.
17. Add 100-200mL of Ultrapure water or 10mM Tris-HCl/1mM EDTA (low salt TE) and store tube at 4C overnight to dissolve pellet or incubate at 65°C.
18. Transfer to new eppendorf and load 2ml on agarose gel to check quality
19. Measure concentration using Spec or fluorometer
20. Store DNA at -20°C for long-term storage or at 4°C for temporary storage