Protocols

0.5M EDTA pH 8.0

Ingredients                                Amount                                      Final concentration [x]

Na₂EDTA·2H₂O                       186.12g

NaOH                                         20g

Note: 

Final volume is 1L.

Adjust pH to 8.0 with NaOH.  NaOH solution is more instantaneous than NaOH pellets.

10% SDS

Ingredients                                Amount                                      Final concentration [x]

SDS powder                             100g

dH₂O                                           900mL

Note:

Dissolve SDS in 900mL of dH₂O, then bring total volume up to 1L.

Be careful not to inhale SDS powder.

10x PCR buffer

Ingredients                                Amount                                      Final concentration [x]

Tris pH 9.2                                             25mL                                          

(NH₄)₂SO₄                                               1.056g                           160mM

1 M MgCl₂                                              750μL                            15mM

1 M MgCl₂                                             1.25mL                            25mM

1 M MgCl₂                                             1 mL                                 20mM

Tween 20                                             500μL                               1%

dH₂O

Note:

Use only ONE of the three concentrations of MgCl_2, not all the concentrations.

Bring up to 50mL, after mixing, with dH₂O.

1M Tris buffer pH 8.0 or 7.5

Ingredients                                Amount                                      Final concentration [x]

Tris                                              121g

dH₂O                                           800mL

HCl (concentrated or 12M)    ~35mL

Note:

Dissolve Tris in the dH₂O, then add the HCl.

Slowly add more HCl until the pH is at 8.0 or 7.5, whichever is desired.

1M Tris buffer pH 8.5 or 9.2

Ingredients                                Amount                                      Final concentration [x]

Tris                                              121g

dH₂O                                           800mL

HCl (concentrated or 12M)    ~20mL

Note:

Dissolve Tris in the dH₂O, then add the HCl.

Slowly add more HCl until the pH is at 8.5 or 9.2, whichever is desired.

7.5M ammonium acetate

Ingredients                                Amount                                      Final concentration [x]

ammonium acetate                  577.9g                                       7.5M

dH₂O                                          700mL

Note:

Dissolve ammonium acetate in 700mL dH₂O, then bring up to 1L. (Final volume 1L).

DNA extraction buffer (pine needle method)

Ingredients                                Amount                                      Final concentration [x]

5M NaCl                                    25mL                                           250mM

1M Tris HCl ph 8.0                  100mL                                          200mM

0.5M EDTA pH 8.0                  25mL                                            25mM

10% SDS 25mL 0.5%

PVP 30g 6%

dH₂O 300mL

Note:

Mix above ingredients, then bring total volume up to 500mL.

Loading Dye

Ingredients                                Amount                                      Final concentration [x]

Glycerol                                      15mL                                         30%

0.5M EDTA pH 8.0                   5mL

dH₂O                                           30mL

bromophenol blue                     50mg

xylene cyanole                           50mg

Note: final volume is 50mL.

20x SB buffer pH 8.0

Ingredients                                Amount                                      Final concentration [x]

dH₂O                                           800mL

NaOH                                         8g

Boric acid                                  50g

Note: 

Mix well until fully dissolved.

Check pH, it should be pH 8.0, if so, raise volume to 1L with dH₂O.  If not, add boric acid as needed, then raise the volume.

10x TBE buffer

Ingredients                                Amount                                      Final concentration [x]

Tris (powder)                            108g

Boric acid                                  55g

0.5M EDTA pH 8.0                  40mL

dH₂O                                          800mL

Note:

Mix well and then bring volume up to 1L, and mix again (final volume is 1L).

Sephadex G50

Ingredients                                Amount                                      Final concentration [x]

Sephadex (powder)                12.5g

QH₂O                                         250mL

Note:

Stir for 5 minutes before each use.

The ratio for this is 50mg Sephadex/mL of water

Final volume 250mL

DNA extraction protocol (Brunner method) 

Brunner et al. (2001) Molecular identification of fine roots of trees from the Alps: reliable and fast DNA extraction and PCR–RFLP analyses of plastid DNA. Molecular Ecology 10, 2079–2087. 

Lyophilized fine roots were ground to a fine powder in a swing mill (Retsch, type MM2000) with two steel balls (4 mm diameter). 

1. Weigh 30 mg of lyophilized, ground fine roots 

2. Add 650 µL of extraction buffer, vortex until well mixed, then incubate for 30 min at 65ºC (in a shaking water bath) 

3. Add 1 volume of chloroform/isoamylalcohol (24 : 1), thoroughly mix, and centrifuge for 10 min at 10 000g 

4. Transfer the top supernatant to a clean tube. The bottom liquid is the chloroform:isoamylalcoho solution. (Repeat steps 3 and 4 again with the supernatant in the new tube). 

5. Precipitate DNA by adding 1.5 volumes of isopropanol and incubating for at least 1 h at -20°C. 

6. Centrifuge for 30 min at 20 000g. 

7. Pour out liquid or pipette out if the pellet of DNA is not stuck to the bottom of the tube. 

8. Wash pellet with 70% chilled ethanol and pour out ethanol or pipette out. Allow all ethanol time to evaporate. 

9. Dissolved in 150 μL TE buffer (10 mM Tris–HCl pH 8.4, 1 mM EDTA) by a 5-min incubation at 65°C.

DNA Extraction Protocol 

Lodhi et al. A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species Plant Molecular Biology Reporter 12 (1): 6-13.)

Solutions needed:  

2. Grind 0.5 g of leaves using mortar and pestle with liquid nitrogen. 

3. Add 5 mL of extraction buffer to the ground leaves and mix in the mortar. 

4. Pour the slurry into clean 15-mL polypropylene centrifuge tubes . 

5. Add 50 mg polyvilylpolypyrrolidone( PVPP) and invert the tubes several times to mix thoroughly with the leaf slurry; the final concentration of PVP is 100 mg/g leaf tissue. 

6. Incubate at 60°C for 25 minutes and cool to room temperature. 

7. Add 6 mL of chloroform-octanol and mix gently by inverting the tubes 20 to 25 times to form an emulsion. 

8. Spin at 6000 rpm for 15 minutes in a tabletop centrifuge at room temperature.

9. Transfer the top aqueous phase to a new 35-mL centrifuge tube with a wide-bore pipette tip. (A second chloroform-octanol extraction may be performed if the aqueous phase is cloudy due to the presence of PVP.) 

10. Add 0.5 volume of 5M NaCl to the aqueous solution recovered from the previous step and mix well. 

11. Add two volumes of cold (-20°C) 95% ethanol and refrigerate (4 to6°C) for 15-20 minutes or until DNA strands begin to appear. (The solution can be left for one hour or more if necessary.) 

12. Spin at 3000 rpm for three minutes and then increase speed to 5000 rpm for an additional three minutes at room temperature. 

13. Pour off supernatant and wash pellet with cold (0 to 4°C) 76% ethanol. Completely remove ethanol without drying the DNA pellet by leaving the tubes uncovered at 37°C for 20 to 30 minutes. 

14. Dissolve m 200 to 300 pL TE. 

15. (Treat with 1 pL RNase A per 100 pL DNA solution and incubate at 37°C for 15 minutes.) 

16. Store DNA at -70°C for long-term or -20°C for short-term storage 

Notes 

1. Avoid thawing before grinding the leaf tissue. Although leaves should be thoroughly crushed before adding extraction buffer, it is important not to grind the leaves into a very fine powder, as it results in shearing of DNA. 

2. This differential spin helps to keep DNA at the bottom of the centrifuge tube.

DNA Extraction (Pine Needle Protocol)

 Reagents

1.   Extraction butter:

a.       250 mM NaCl

b.      200mM Tris-HCl pH8.0

c.       25 mM EDTA

d.      0.5% SDS

e.       6% w/v PVP

2.      2. 7.5 M Ammonium Acetate

3.      3. Isopropanol

4.      4. 70% Ethanol

Procedure

1.            1.  Grind pine needles (~3-5 3mm section) in 1.5 ml tube in 500 μl extraction buffer using a plastic pestle attached to a hand held drill.

Note: Can use other method to grind leaves.

2.      Put tubes in heat block set at 95⁰C for 20 min (open tops briefly, then close after putting in heating block to reduce initial pressure) – invert tubes after 10 min to mix samples. (Vortex)

3.      Let samples cool to room temperature, then add ¹/₂ volume of 7.5 M Ammonium Acetate (250 μl) and shake or vortex vigorously for ~10 seconds.

4.      Put samples on ice or freezer for 10 min.

5.      Centrifuge at full speed for 10 min to pellet leaf material.

6.      Pour supernatant in a clean tube – if pellet is loose and it is difficult to avoid pouring out parts of the pellet then centrifuge supernatant in a clean tube to pellet remaining particles and then remove supernatant again and place in new tube.

7.      Add 0.7 volumes of isopropanol and mix by inverting the tubes for ~15 seconds (you can weigh a few samples to determine the average amount (μls) of supernatant you poured out in step 6).

8.      Spin at a full speed for 15minutes to pellet DNA

9.      Pour off isopropanol and invert on paper towers

10.  Wash pellet with 400 μl of 70% ethanol, spin for 5 min, pour off alcohol and air dry.

11.  Resuspend DNA in 100 μl 10 mM Tris-HCl pH 8.5 overnight

12.  Vortex tubes briefly then spin at full speed for 3 min – if a pellet is visible remove buffer (with resuspended DNA) and place in a clean tube.