Simultaneous extraction of chloroplast and nuclear DNA

Alfred E. Szmidt and Xiao-Ru Wang

  1. Put foliage in a metal pot, pour some liquid nitrogen and crash to small pieces with a mortar rod. For good cpDNA yield we recommended to use at least 15 g of foliage. For nDNA or total DNA much smaller amounts of material can be used.
  2. Transfer immediately deep frozen material to a coffee grinder and grind for 10 - 30'' to a fine powder (it is important that the powder should not melt during grinding); transfer powder to a plastic beaker and stir occasionally with a plastic spatula; allow to reach app. 5ºC. If you work with small amount of material (below 5 g), transfer powder to a mortar and allow to reach app. 5ºC.
  3. Add 350 ml/100 g foliage of extraction solution E-1, stir with a plastic spoon for 5-10'. Shake well extraction solution E-1 before use! . If you use a mortar, grind powder to a fine pulp with a rod.
  4. Filter homogenate through 5-10 layers of cheesecloth.
  5. Filter through 1 layer of Miracloth®, Calbiochem (soak Miracloth® in distilled water before use); do not squeeze too hard.
  6. Transfer filtrate to 50 ml Sorvall® tubes and centrifuge at 3000 rpm for 2.5' (SS-34 rotor, Sorvall®).
  7. Drain pellet and wipe tube inside with Kleenex®.
  8. Suspend pellet using nylon paint brush (No. 8) in 40 ml wash solution E-2.
  9. Centrifuge for 2.5' at 5000 rpm (SS-34 rotor Sorvall®).
  10. Drain pellet, wipe tube inside with Kleenex® and repeat p. 9 one more time.
  11. Suspend pellet with a paint brush in 5-10 ml of wash solution E-2.
  12. Load suspension on a sucrose step gradient (thawed overnight); use 5 ml suspension per tube.
  13. Centrifuge for 20-30' at 3000 rpm (HB-4 swing-out rotor, Sorvall®).
  14. Collect chloroplast band into 50 ml Sorvall® tube and make to 40 ml with PTE- buffer, suspend carefully with a paint brush. Collect nuclear pellet into 50 ml Sorvall® tube and make to 40 ml with PTE- buffer. Suspend carefully with a paint brush.
  15. Centrifuge both fractions at 5000 rpm for 3' (SS-34 rotor Sorvall®).
  16. Drain chloroplast and nuclear pellets and wipe tubes inside with Kleenex®.
  17. Repeat p. 15-16, 3-4 times (2 times are enough for nuclear fraction). Chloroplast pellets may become quite hard, suspend them carefully with a paint brush after each centrifugation.
  18. Suspend separately chloroplast and nuclear pellets in 2.5 ml of freshly prepared Proteinase K solution in PTE buffer (1 mg/ ml).
  19. Transfer chloroplast and nuclear suspensions to calibrated 10 ml plastic tubes with stoppers and keep for 30' at room temperature.
  20. Add rapidly 60 µl / tube of cooled 10% Triton X-100 in PTE buffer and keep for 1-2 hrs at room temperature to aid lysis.
  21. If more then 50 g of foliage was used: spin down nuclear lysate for 10' in a table centrifuge and collect supernatant. Skip this step for chloroplast fraction. Skip it too for nuclear fraction obtained from a small amount of material since much of DNA is left in the pellet.
  22. Prepare CsCl gradients.

Solutions

Extraction solution E-1

0.5 M Stock Solution

Dissolve the following components in app. 800 ml of distilled water.

  1. 0.3 M sucrose (102.69 g)
  2. 10% polyethylene glycol (PEG 8000 - Sigma®) (100 g)
  3. 25 mM HEPES (5.96 g)
  4. 2 mM EDTA III 0.7 g (4 ml)
  5. 1 mM CaCl2 110 mg (2 ml)
  6. adjust pH to 6.7 with 10 M NaOH
  7. make up to 1000 ml with distilled water
  8. add 1 g of bovine serum albumin (BSA, fraction V, Sigma®)
  9. add 6 g of Polyclar AT (Serva®)
  10. add 78 µl of 2-mercaptoethanol (=10 mM)
  11. Store at -20ºC in a plastic bottle. Melt down overnight at 4ºC before use.

Wash Solution E-2

0.5 M Stock Solution

Dissolve the following components in app. 800 ml of distilled water

PTE buffer (50 mM Tris-HCl, 10 mM EDTA, pH 7.8)

Dissolve the following components in app. 800 ml of distilled water.

Sucrose gradient solutions and preparation

Stock solution

Dissolve the following components in app. 200 ml of distilled water.

Gradient solutions

20% 30% 40% 50% 60%
Sucrose (g) 20 30 40 50 60
Stock solution (ml) 40 40 40 40 40
Distilled water: make up to 100 100 100 100 100
Store at -20ºC

Step gradient preparation

  1. add sequentially to a 50 ml Sorvall® tube the sucrose solutions described above: 60% - 7.5 ml; 50% - 7.5 ml; 40% - 7.5 ml; 30% - 7.5 ml; 20% - 3 ml
  2. freeze prepared gradients at -20ºC overnight
  3. thaw overnight at 4ºC a day before use

50x TE buffer for dialysis (500 mM Tris/HCl, 50 mM EDTA)

Dissolve the following components in app. 800 ml of distilled water

General comments

  1. Please note that plastic ware is used in all steps - this is to avoid DNA shearing.
  2. We used this protocol for more than 120 species of Pinus and Picea with very good results. We tried it also quite successfully on species from all other gymnosperm genera except Gingko. For some species it may be necessary to eliminate PEG from E-1 and E-2 solutions to avoid formation of rubber-like pulp after addition of E-1 (step 3).
  3. It is preferable (but not absolutely necessary) to collect material after cessation of growth i.e. when the starch content is low.
  4. Raw material collected in the field can be stored at - 20ºC for more than 5 years in sealed plastic bags.
  5. If followed by CsCl gradient purification this protocol always gives digestible DNA, but the degree of purity of cpDNA may vary among species.
  6. It is possible to replace purification using CsCl gradient with e.g. phenol extraction followed by ethanol precipitation. It reduces the costs but may yield undigestible DNA.

by Alfred E. Szmidt