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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.4/2.3


TITLE: BVDV DETECTION BY USE OF A DiG LABELLED PROBE FOLLOWING RT PCR


PROCEDURE

RT-PCR

  1. Carry out RT-PCR on selected samples using a known positive and negative sample as controls as per Protocol AHC/1998/3/3.4/2.2.

Electrophoresis and Denaturation:

  1. Run the following on an agarose/TBE buffer gel (1 to 1.5% agarose gel used as standard).
  2. - DNA product: 30ml

    - 10ml of 10ng/ml molecular weight markers digoxigenin labelled (Boehringer or equivalent)

    - positive control: unlabelled probe if possible (1/1000th of what you would normally run to see)

    Run gel until 30mm from the end. Stain in ethidium bromide (in the electrophoresis tray if possible) and photograph.

  3. Submerge the gel to effect denaturation in 100ml of denaturing solution for 30 minutes changing the solution after 15 minutes. Incubate on a rotary platform. N.B.: This time may be altered depending on the % of agarose and thickness of gel; 1% and medium size gel = 20 minutes. The bromophenol blue goes pale and slightly translucent when the denaturation has been effected.
  4. Briefly submerge the gel in distilled water to effect rinsing.
  5. Submerge the gel to effect neutralisation by soaking for 30 minutes in 100ml neutralising solution at RT on a rotary platform. Change the neutralising solution after 15 min.

Southern Blot:

  1. Cut positively charged nylon membrane the same size as the gel. Handling of the membrane throughout should be minimal and only with blunt ended forceps and unpowdered rubber gloves at the edges.
  2. The following pieces are cut out of Whatmann 3mm filter paper:
  3. - 6 pieces of same size as the gel

    - 2 wicks the same width as the gel and long enough to safely dip into the reservoir.

  4. The set up of the blot is shown below:
    All equipment used should be ultra-clean
  5. (i) Place the tray, support and glass plate in situ. Fill the tray with 20 x SSC.

    (ii) Wet the wicks in 2 x SSC and then drape across the top of the glass plate. Roll out air bubbles with a glass pipette.

    (iii) Wet 2 of the filter papers in 2 x SSC and position on top of the gel. Remove air bubbles.

    (iv) Place the inverted gel (underside uppermost) taking care to exclude air bubbles.

    (v) Soak membrane in 2 x SSC for 2 minutes and place on top of the gel. Remove air bubbles. Orientate the filter by removing the top right hand corner.

    (vi) Soak 2 further filter papers in 2 x SSC and place on top followed by the final 2 dry pieces.

    (vii) Place four strips of autorad film round edges of the gel to stop short circuiting.

    (viii) Place a whole box of tissues on top, taking care to misalign the central seam gap of the tissues by folding. Place a heavy weight (500gm) on top.

  6. The transfer is allowed to continue overnight. Remove the top levels of the blot and flip the gel and membrane over onto Whatmann paper. Mark the wells with a soft pencil. Place the membrane on a UV transilluminator, l254nm. Transilluminate the membrane, 3 minutes on each side (the side to which the DNA transfer was effected being first). Air dry for 10 minutes. Can store wrapped in filter paper and then foil, in a dry cupboard at room temperature if desired.

Prehybridisation and Hybridisation

  1. Submerge membrane in 20ml of Easy Hyb in a sealed box. Place on a rotary shaker in a preheated hybridisation oven at 68oC 1C for 1 to 5 hours.
  2. Remove the pre-hybridisation buffer and save in a Universal (can be used up to three times). Add 20ml pre-hybridisation buffer with 30 to 50 ng probe added. The probe must be boiled for 4 minutes and iced for 5 minutes before adding to the pre-hybridisation buffer. If the probe/mix is already made up then boil the whole mix as described for the probe. Hybridise overnight at 68oC 1C.

 High Stringency Washes:

  1. Pour off and save the probe mix into a plastic Universal (store at +2 to 8oC).
  2. Wash the membrane twice with 50ml 2 x SSC + 0.1% SDS (100ml: 1ml 10% (w/v) SDS + 10ml 20 x SSC 89ml distilled water) for 5 minutes at room temperature. Warm this solution at 37oC if the SDS comes out of solution.
  3. Wash the membrane twice with 50ml 0.1% SSC + 0.1% SDS (100ml: 1ml SDS + 0.5ml 20 x SSC) for 15 minutes at 68oC 1C.

Note these quantities are for a 100cm2 membrane. More may be needed of larger membranes are used.

Chemiluminescent Detection

Steps 1-8 may be done at room temperature in hybridisation bottle on a spiramix or in a box on a rotary shaker (preferred method).

  1. Wash the membrane for 5 minutes in 100ml washing buffer (200ml: 600ml Tween 20 [0.3% (w/v)] + 199.4ml Maleate buffer). Remove.
  2. Incubate in 100ml 2% blocking buffer (100ml: 30ml 10% blocking buffer + 70ml Maleate buffer) for 1 hour. Pour off and discard the buffer.
  3. Dilute the anti-digoxigenin-AP fragments 1:10,000 in 2% blocking buffer (2ml fragments + 16ml Maleate buffer + 4ml 10% blocking buffer). Incubate the membrane in a dedicated container, without shaking, for 30 minutes in 20ml of this dilution. Pour off and discard.
  4. Remove unbound conjugate by washing, in a new container, with shaking, with 100ml washing buffer (see '1') for 15 minutes twice. Pour off and discard.
  5. Wash for 5 minutes in Maleate buffer.
  6. Equilibrate, with shaking, in 20ml buffer 3 (10ml 10 x Tris HCl (pH 9.5)/NaCl mix + 5ml 20 x MgCl2 + 85ml distilled water) for 15 minutes. Pour off, discard and repeat with fresh buffer for a further 15 min.
  7. Add 10ml of 1 in 100 dilution of CSPD in buffer 3 (100 ml CSPD in 9.9ml buffer 3) to the membrane for 1 to 5 minutes (maximum or the blot is overstained). Pour off and save in a foil wrapped Universal at 2 to 8oC - can re-use twice (N.B.: if the CSPD is exhausted wash the membrane twice in distilled water for 5 minutes, re-equilibrate in buffer 3 and apply fresh CSPD).
  8. Remove the membrane and allow excess liquid to drain onto a dry Whatman filter. Blot for a few seconds and then place face down in a plastic bag. Heat seal the bag, working rapidly to prevent the filter drying out. Wrap up completely excluding air bubbles.
  9. Pre-incubate for 5 to 15 minutes at 37oC 1C.
  10. Lay in a small cassette holder DNA side uppermost. Add orientated film and expose for 15 to 30 min at RT.
  11. Develop film for 2 minutes in the developer, submerge in stop solution for 1 minute and then fix for 1 minute. Rinse in tap water for 2 to 3 min and air dry.
  12. To save the membrane store at +2 to 8oC in 2 x SSC in a box, or in a sealed hybridisation bag with 2 x SSC in it. Do not allow the membrane to dry out.

Removal of Probe

  1. Rinse the membrane twice in distilled water for 10 minutes twice with shaking to remove the CSPD.
  2. Incubate the membrane in 100ml 0.4M NaOH, with shaking, at 45oC 1C for 30 minutes.
  3. Transfer to 100ml of 0.1 x SSC + 0.1% 0.5% (w/v) SDS + 0.2M Tris-HCl pH7.5. (500ml 20 x SSC, 1ml 10 x SDS, 20ml 1M Tris HCl pH7.5). Incubate at 45oC 1C for 15 minutes with shaking.
  4. Storage is effected at 2 to 8oC in a stomacher bag in 2 x SSC with all the air bubbles removed. Storage should only be effected of stripped membranes.

Quantification of Labelled Probe

  1. Prepare serial dilutions of the probe and of a labelled control DNA (with a known concentration of DNA).
  2. Spot 1ml of each of the dilutions onto a dry positively charged nylon membrane. Air dry and then or UV crosslink for 3 minutes (1 min each side) at l254nm.
  3. Follow steps 2 to 12 as in the chemiluminescent detection.
  4. Compare the spot intensities of the control and experimental dilutions to estimate the concentration of the experimental probe.

REAGENTS

  • 20 x SSC

Sodium chloride 175.3g

Sodium Citrate 88.2g

Distilled water 800ml

Adjust to pH 7.0. Make up to 1l with distilled water. Dilute 1/1000 and check conductivity (around 45mS). Autoclave according to Protocol 942/-/-/223 and store at room temperature.

  • Maleate buffer (1l):

Maleic acid 11.61g

NaC1 8.766g

Distilled water 800ml

Dissolve. Adjust pH to 7.5 with 5M NaOH. Add 35ml and then add drop by drop carefully as this solution easily over pH's. Adjust final volume to 1l. Store at room temperature.

  • Washing buffer (1l):

Tween 20 3ml

Maleate buffer to 1l

  • 10% Blocking Buffer (store at 2 to 8oC):

Blocking reagent 50g

Maleate buffer to 500ml

Heat at 68oC 1C for 1 hour to completely dissolve. Autoclave and store at +2 to 8oC.

  • Buffer 3:

A precipitate tends to form in this buffer if the Mg2+ is added too early. Therefore stock solutions are made up and autoclaved. Buffer 3 is made freshly from these as needed.

1l

10x Tris-HCl pH9.5 121.1g (1M)

10x NaC1 58.44g (1M)

20x MgCl2 203.3 (1M)

For 100ml of buffer 3 add 10ml of 10 x Tris HCl/NaCl mix + 5ml 20 x MgCl2 to 85ml of sterile distilled water in a sterile bottle.

Denaturing Solution

20g NaOH

87.66g NaC1

Distilled water to 1l

Autoclave. Store at room temperature.

Neutralising Solution

60.55g Tris pH 7.4

87.66g NaCl

Distilled water to 1l

Autoclave. Store at room temperature.

10% SDS

10g SDS to 100ml distilled water

Heat to approximately 68oC in waterbath for 2 hours. DO NOT AUTOCLAVE. Store at room temperature.


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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