Benton Davis Blots

Day 1

Prepare in advance:

Chill plates containing bacteriophage-lysed E. coli for at least 1 hour at 4℃ (2 hours works well).

Use 2 nitrocellulose filters for primary screening, and 1 filter after that per Petri dish (S & S BA85 or Amersham Hybond-C 0.45 µm nitrocellulose; 132 mm diameter for 150 mm Petri dishes, or 82 mm diameter for 100 mm diameter petri dishes). Handle these as little as possible and only by the edges using gloved hands and forceps. Number each filter with India ink or pencil to correspond with the number of each respective plate.

25 G needle and small ｄｒｏｐ of India ink on piece of parafilm.

Tray with base solution .

Whatman 3MM filter paper or gel blot paper to blot filters dry after removal from base and neutralizing solution.

Tray with neutralizing solution.

Whatman #1 filter paper to layer between filters.

80℃ vacuum drying oven (preheated 1 hour).

Hybridization buffer (sterile 5x SSPE; 10x filter sterilized Denhardt's solution; sterile 0.1% SDS; use sterile H2 O - do not autoclave hybridization buffer; warm to 65℃ before use).

Container, or bag with heat sealer, for hybridization (we have used one Kapak/Seal-a-Meal bag for as many as 40 filters on a given day).

2x SSPE (30-50 ml) diluted in water from a 20x SSPE stock. Protocol:

Slowly lay nitrocellulose filters on the agar surface from the center to the side. Avoid trapping bubbles between filter and agar . Handle filters only by their edges with forceps. Mark position of filter on the plate by piercing the filter with a 25G needle dipped briefly in India ink in three asymmetrically positioned spots per filter.

For primary screenings, blot one filter on each petri dish for one minute and the second filter for five minutes on the same plate. After primary screening, blot each dish with only one filter for two minutes.

Remove filters from the petri dishes with flat-end tweezers, lifting from one edge and rolling the filter off the agar. Avoid tearing filter and lifting agar from the surface. Chilled plates help to avoid the latter problem.

Place filter in base solution for 1 minute, agar side up. The base solution (and the neutralizing solution below, may be used to saturate a Whatman 3MM filter paper, and the nitrocellulose filters laid on this, rather than floating freely in a container of each solution.

Blot filters on dry Whatman 3MM or gel blot paper to remove excess base.

Transfer filters to neutralizing solution for two minutes.

Blot filters on dry Whatman 3MM paper and stack between Whatman #1 filter paper.

Dry in 80°C vacuum oven 1-2 hours. No moisture should be on the glass oven window. It takes about 2 hours for the large filters and an hour for the small filters. After baking, nitrocellulose membranes are very fragile.

Before hybridization, wet filters briefly in 2x SSPE and stack filters on top of one another. (You can omit this step if you have only a few filters to hybridize. This pre-wetting step helps reduce bubbles which may become trapped between filters when large numbers of filters are added to a bag.)

Allow excess solution to drip from the filters and transfer to Kapak bags.

Add hybridization buffer (preheated to 65℃) to wet filters and remove air bubbles within bag. Ten ml of buffer per bag is usually sufficient for a few (about 6) filters.

Heat seal bag and allow to warm in 65℃ oven for 15 minutes.

Denature the radiolabelled DNA probe (1-2 x 106 cpm/filter) at 95℃, 5-10 minutes.

Add probe at corner of sealed bag using a 25G needle and syringe. Heat seal below the injection site. Briefly rinse corner of bag around injection site to remove any probe on outside of bag.

Hybridize the filters at 65℃ in a slowly rotating H 2 O bath overnight.