***Populus* nuclear DNA extraction protocol**[^1]

***Reagents***

β-mercaptoethanol

Bovine Serum Albumin (BSA)

Cesium Chloride

EDTA, 0.5 M, pH 8.0

Ethanol, 100%

Ethidium bromide, 1 mg/ml

NaCl, 5 M

2-(N-Morpholino)ethanesulfonic Acid (MES)

NP-40

Percoll

Sarkosyl, 10% (syn = n-lauroylsarcosine or sodium lauroyl sarcosine)

Spermidine trihydrochloride

Spermine tetrahydrochloride

Sucrose

10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (TE Buffer)

TE-saturated isoamyl alcohol

***Equipment and supplies***

18-gauge needles

3 cc syringes

Centrifuge tubes, 50 ml polypropylene

Centrifuges (Beckman and Sorvall Ultra) and rotors (GH 3.8 & SW 41) at 4
ºC.

Cheesecloth (8x8 in squares, autoclaved)

Dounce homogenizers

Funnels

Ice

Mortar and pestle

Miracloth (8x8 in squares, autoclaved)

Polyallomer tubes

Transfer pipets

Waring Blender glass top at 4 ºC.

***Solutions***

**Nuclear Isolation Buffer (NIB):**

  -------------------- ---------------- ----------------- -----------------
  Reagent              weight           5X                1X (final)

  Sucrose              85.6 g           1.25 M            250 mM

  NaCl                 2.0 ml 5M        50 mM             10 mM

  MES                  2.12 g           50 mM             10 mM

  EDTA                 10.0 ml 0.5 M    25 mM             5 mM

  Spermine (mw=348)    52.0 mg          0.75 mM           0.15 mM

  Spermidine (mw=255)  128.0 mg         2.5 mM            0.5 mM

  BSA                  n/a              n/a               2.0 %
  -------------------- ---------------- ----------------- -----------------

Add sterile R.O. distilled water to 200 ml.

Sterilize by filtration and store at 4 ºC.

Before use:

Dilute 100 ml 5X NIB to 480 ml 1X with cold sterile R.O. distilled
water.

Add NP-40 to 0.6% (3.0 g).

Add β-mercaptoethanlol to 2.0% (10.0 ml).

Adjust final volume to 500 ml.

Chill thoroughly before use.

**Floating Buffer** (\~50 ml):

  ----------------------------------- -----------------------------------
  Reagent                             weight

  β-mercaptoethanol                   1.0 g

  5X NIB                              6.0 g

  Percoll                             45.0 g

  NP-40                               0.3 g
  ----------------------------------- -----------------------------------

Prepare immediately before use.

**Proteinase K Buffer :**

  ----------------------- ----------------------- -----------------------
  Reagent                 volume                  \[Final\]

  Proteinase K (20 mg/ml) 500 μl (or 10 mg)       1 mg/ml

  EDTA (0.5 M)            400 μl                  20 mM

  Sarkosyl (10%)          1.0 ml                  1%

  β-mercaptoethanol       1.0                     2%

  TE pH 8.0               To 10 ml                
  ----------------------- ----------------------- -----------------------

**Personal Protective Equipment**

1\. Nitrile gloves for Ethidium bromide use.

2\. Lab coat

3\. UV eye protection

4\. Read all pertinent MSDS sheets

**Safety, Health and Chemical Hygiene Requirements for Conducting this
Protocol:**

1\. Ethidium bromide is a powerful mutagen and a moderately toxic
irritant and sensitizer. Gloves must be worn when working with the CsCl
gradients containing highly concentrated solutions of EtBr. After each
handling of solutions containing EtBr, contaminated gloves must be
discarded into a clean plastic bag and disposed of in the waste
container, to prevent the spreading of EtBr to adjacent areas of the
work bench. Perform syringe extractions in a dedicated, reusable
container to catch any EtBr spills.

2\. The following must be collected and disposed of as Hazardous wastes:
Ethidium Bromide/isoamyl-alcohol; CsCl/EtBr gradient.

3\. Ultraviolet (UV) radiation is dangerous to exposed skin, and
especially the eyes. Always wear UV eye protection.

4\. Dispose of absorbent bench pad in EtBr solid waste if contaminated.
Check benchtop with hand held UV light, and clean up any EtBr spills
with soap and water.

5\. Use isoamyl alcohol in a fume hood. Wear nitrile gloves and eye
protection.

***Protocol***

1\. Powder 5 g young leaves or 20 gram fresh or frozen root tips in
mortar and pestle using N~2~(l). Add N~2~(l) periodically to keep tissue
frozen. Add to 200 ml 1X NIB on ice and mix by stirring.

2\. Pulse in cold Waring blender 15 s; filter through 4 layers
cheesecloth + 1 layer Miracloth into 4-50 ml centrifuge tubes.

3\. Centrifuge at \~2,000 x g (4K rpm) for 10 min at 4˚ C in Beckman
GA10 fixed angle rotor to obtain crude nuclear pellet.

4\. Discard supernatant. Resuspend pellets in 40 ml 1X NIB and repeat
#3.

5\. Discard supernatant. Resuspend pellets in 40 ml Floating Buffer
using Dounce homogenizer. Aliquot equal weight into 4 pollyallomer tubes
and centrifuge at 5000 x g (6400 rpm) 30 min at 4˚ C in SW41 rotor.

6\. Remove floating nuclei with transfer or Pasteur pipette and
resuspend in 10.0 ml 1X NIB with Dounce homogenizer on ice. Centrifuge
at \~2,000 x g (4K rpm) for 10 min at 4˚ C in Beckman GH3.8 rotor to
pellet nuclei.

7\. Add 5 ml 1 mg/ml proteinase K solution and incubate with gentle
shaking (50 rpm) at 37 ºC overnight to lyse nuclei.

Use the following protocol (a) for ultrapure nuclear DNA

8a. Centrifuge at 5000 x g (6400 rpm) 15 min at 4˚ C in Beckman GA10 to
remove nuclear membrane debris.

9a. Add 1.0 g CsCl to each ml supernatant and adjust density to ρ=1.55
(by weight). Add ethidium bromide to 200 μg/ml. Place in 2 polyallomer
tubes and fill to top with mineral oil.

10a. Centrifuge in SW41 at 100,000 x g (28.5 K rpm) for 44 h at 16 ˚C.

11a. Recover the band with an 18 gauge hypodermic needle (as described
in Sambrook et al. 1989 p. 1.43-1.45).

12a. Remove ethidium bromide by adding an equal volume of TE-saturated
isoamyl alchohol. Mix phases by vortexing. Centrifuge at 1,500 rpm in
benchtop centrifuge 3 min at 25˚ C. Transfer lower, aqueous phase to
clean tube and repeat 3-4 times until all pink color is removed from
both phases.

13a. Dilute with 3 volumes TE and precipitate 2 volumes of ethanol
(total final volume is 6X original). Mix for 15 min and store at --20 ˚C
(o/n) to precipitate CsCl. Remove DNA solution to new tube and
centrifuge at 5,000 x g for 30 min at 4˚ C. Dissolve DNA pellet in
appropriate volume TE pH 8.0.

14a. Quantitate with spectrophotometer.

15a. (optional) Mix 10 μl DNA by suspending in 90 μl 1% liquid LMP
agarose. Mix and pipet into plug mold. Cool and place in 1% Sea Plaque
in 0.5X TBE pulse field gel using yeast chromosome PFGE marker. Run gel
24 h, 90s, 6 V/cm, 120 ˚, 14 ˚C.

Use the following for crude nuclear DNA:

8b. Add equal volume chloroform:isoamyl alcohol (24:1). Mix 10'

9b. Centrifuge 20 min at 10,000 x g in SS 34 at 4˚ C.

10b. Remove aqueous phase. Repeat chloroform extraction

11b. Add TE to aqueous phase to 10 ml. Precipitate with 2X 100% ethanol.

12b. Centrifuge 20 min at 10,000 x g in SS 34 at 4˚ C.

13b. Wash pellet in 70% ethanol

14b. Transfer to 1.5 ml tube. Centrifuge 20' 10,000 x g 4˚ C.

15b. Dry in speed vac 3'. Resuspend in 500 μl TE.

[^1]: Adapted from Weising K, H Nybom, K Wolff and W Meyer. 1995.
    Chapter 4: Methodology. *In DNA Fingerprinting in Plants and Fungi*.
    Boca Raton, FL: CRC Press. Pp. 57-61.