BOT/MBIO/ZOO 5364

Preparation of DNA for Transmission Electron Microscopy

Stock Solutions for Mounting DNA

• Parlodion 3.5% w/v in n-pentyl acetate.  Add molecular sieves to dehydrate.  It takes about 3 days for the parlodion to dissolve.
• Uranyl acetate 5 x 10^-2 M in 50 mM HCl (store at 4°C).
• Cytochrome c 1 mg/ml in 1 mM EDTA.  Filter (0.2 µm).  Store at 4°C.

Stock specific to AQUEOUS technique:

• 5 M ammonium acetate pH 7.5, 10 mM EDTA (store at 4°C)

Stocks specific to FORMAMIDE technique:

• 1 M Tris pH 8.5, 0.1 M EDTA
• Isopentane (2 methyl butane)
• Ethanol 90%
• Dichromic acid cleaning solution:            Sodium dichromate                     100 g

                                                                        Sulfuric Acid                               100 ml

                                                                        H₂O                                      to 1000 ml

(This solution is used to clean the glass slides.  Rinse off the acid solution with distilled water and briefly soak the slides in 0.25 M NH₄ acetate before use.)

• Acetone
• 0.25 M NH₄Ac, filter and refrigerate.  This is used to rinse slides.
• Formamide.  Check the pH before using.  If the pH is <7, mix the formamide with a mixed bed resin to deionize the solution (BioRad AG501-X8 mixed resin bed).  Filter the deionized formamide through a paper filter and store at -20°C.

Preparation of Grids

• Rinse grids (200 mesh) in acetone to clean them.
• Lay the grids on a fine screen under clean distilled water.
• Put a drop of the parlodion solution on top of the water.
• The parlodion will spread and begin to wrinkle when it has dried.
• Do not breathe on the film while it is drying, the moisture in your breath will create holes in the parlodion film.
• The film can be deposited on the grids by lifting the screen or by draining the water.
• Blot up excess water from the bottom of the screen with Kimwipes.
• Punch out the grids.
• Allow 2 to 3 hours for the grids to dry before use.
• The grids are good for about 2 days.

The parlodion solution consists of 3.5% w/v parlodion in n-pentyl acetate.  It takes about 3 days for the parlodion to dissolve.

AQUEOUS TECHNIQUE

Spreading Solution:

0.5 µg/ml of DNA

0.1 mg/ml cytochrome c (Sigma Type IIA)

0.5 M Ammonium acetate (NH₄Ac)

1 mM EDTA

It is important to add the constituents in the proper order.  NEVER add a small volume of DNA to a large volume of distilled water as this will denature the DNA.  DO NOT add the cytochrome c to a solution of very low ionic strength since this may cause the cytochrome c to aggregate.

We routinely mix the spreading solution components in the following order:

1.         DNA in an appropriate volume to give about 0.5 µg/ml final concentration (less than 80 µl).

2.         10 µl of NH₄Ac (5M), EDTA (10 mM).

3.         10 µl or less of cytochrome c (1 mg/ml).

4.         Distilled water to give a final volume of 100 ml.

Hypophase:

0.25 M NH₄Ac

• A 50 µl sample of spreading solution is applied to the glass slide at a distance of 1 cm above the slide-hypophase boundary.  We use a 50 µl Hamilton syringe equipped with thin tygon tubing and deliver the sample with a back and forth motion on the slide.  After the sample has run down the slide, the DNA is picked up from the surface of the hypophase with a grid (Parlodion side down) as in Figure 1.

• The grid is shaken in 5 ml of uranyl acetate (5 x 10^-4 M) for 30 sec.  The uranyl acetate stock is diluted into 90% ethanol.  The stained grid is then dehydrated by shaking it in isopentane for 10 sec.

• The grid can now be viewed in the EM or can be shadowed with Pt/Pd to increase the contrast.  Double stranded DNA prepared by the Aqueous technique is well spread while single stranded DNA appears very knotted and collapsed upon itself.

FORMAMIDE TECHNIQUE

Spreading Solution:

0.5 µg/ml of DNA

0.02 - 0.1 mg/ml cytochrome c (Sigma type IIA)

0.1 M Tris pH 8.5

10 mM EDTA

40% v/v formamide

As in the Aqueous technique, the constituents must be added in the proper order.

1.         DNA in an appropriate volume to give a final concentration of 0.5 µg/ml.

2.         10 µl of Tris (1M), EDTA (0.1M)

3.         2 - 10 µl of cytochrome c (1 mg/ml)

4.         Distilled water to bring the volume to 60 µl

5.         40 µl of formamide (deionized formamide is the best).

The spreading solution must be used within 2 hours.

Hypophase:  (100 ml)

10 mM Tris pH 8.5

1 mM EDTA

10% formamide

1.         The hypophase will tend to become acidic so use it within 5 minutes of adding the formamide.

2.         Apply a 50 µl sample of spreading solution to the slide-hypophase boundary.  Wait one or two minutes before picking up the DNA from the hypophase surface with a grid (parlodion side down).  Do two grids.  This will allow the singe stranded structures in the DNA to unravel and become fully extended.

3.         The grid is then stained and dehydrated as described for the Aqueous technique.

4.         The presence of formamide reduces the contrast of stained grids.  We always shadow formamide treated DNA with Pt/Pd (80%/20%).  Wrap 1.5 inches of Pt/Pd wire (.008 in. diameter) around 2-2.5 inch piece of Tungsten wire (.02 in. diameter) that has been bent to form a "V".

5.         The grids are placed on a rotating platform.  The Pt/Pd is connected to the electrodes of the shadower so that it is 8 cm from the grids and 1 cm above the grids.

6.         Double stranded DNA in formamide treated samples is well spread out.  Single stranded DNA is extended but is thinner and kinkier than double stranded DNA.

Technique for Casting DNA

Hamilton syringe                            Tygon tubing

                       Glass slide

  Hypophase

                                                             Teflon bars