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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1.2.3 Appendix 8

TITLE: NON ISOTOPIC CHEMILUMINESCENT ENHANCED PROBE HYBRIDISATION, STRINGENCY WASHES AND DEYBRIDISATION

INTRODUCTION

This procedure describes preparation of Southern blots hybridised with NICETM probe 33.15 (Cellmark Diagnostics) ready for autoradiography. It also describes dehybridisation of these blots and their subsequent storage.

PROCEDURE

  1. Preheat hybridisation oven, sterile water, prehybridisation buffer and wash solution 1 to 49-51oC. Spray the interior of 2 containers (sandwich boxes) with 70% alcohol, wipe dry with tissue and rinse with hot sterile water.
  2. Prehybridise up to 10 Southern Blot membrane by wetting in 1 x SSC, and placing DNA side down in 500 ml of prephybridation buffer at 49.5 to 510C in a sandwich box and agitating for at least 20 minutes.
  3. a) For 10 membranes - add 160ml of hybridisation buffer at 49.5 to 51oC to a hybridisation chamber or sandwich box and add the whole contents of one NICETM probe vial, supplied by the manufacturer

    4. b) For less than 10 membranes - for each membrane to be hybridised, allow 16ml of hybridisation buffer and 10µl of NICETM probe

  4. Transfer the membranes, DNA side down, to the hybridisation buffer plus probe, ensuring there are no air bubbles and handling them with forceps. In the case of single membranes, seal into a polythene bag (internal surfaces sprayed with 70% alcohol and wiped dry with tissue) using Hulme Martin, or equivalent, bag sealer. Cut off one corner of the polythene bag and introduce the hybridisation buffer plus probe onto the DNA side of the membrane. Smooth using a ruler or 10ml pipette to remove air bubbles. Seal the hole using the bag sealer. If any air bubbles remain, make a small hole in another corner, squeeze out bubbles and reseal. Agitate for at least 20 minutes at 49.5 to 51oC.
  5. Wash the membranes by individually transferring each of them DNA side down to 500ml of prewarmed (50 to 51oC) wash solution 1. Agitate for 10 to 15 minutes at 49.5 to 51oC.
  6. Repeat step 5 with fresh wash solution 1.
  7. Wash the membranes by individually transferring each of them DNA side down to 500ml of wash solution 2. at room temperature. Agitate for 5 to 10 minutes at room temperature.
  8. Repeat step 7 with fresh wash solution 2.
  9. Place each membrane DNA side up on a glass or perspex plate which has been sprayed with 70% alcohol and wiped dry with tissue, transfer to a fume cabinet.
  10. Apply approximately 3 to 4 ml of Lumi-PhosTM530 (Cellmark Diagnostics) evenly to each membrane. Cover the whole of the membrane, but do not oversaturate.
  11. Sandwich the sprayed membranes between two 21 x 21cm transparent polyester sheets which have been sprayed with 70% alcohol and wiped dry with tissues. Using a ruler or clean pipette squeeze out any excess Lumi-PhosTM530. Avoid getting Lumi-PhosTM530 on the outside of the sheets. Wipe external surfaces of sheets with tissue wetted with 70% alcohol.
  12. Secure the membrane / polyester 'sandwich' with a small piece of sticky tape ie. Sellotape, along each edge and place in a light proof cassette DNA side against an X-ray sensitive film (eg. Fugi XR). Intensifying screens are not required.
  13. Place the cassette in an incubator at 30 to 37oC and develop the film according to protocol AHC/1998/3/3.1/2.3 Appendix 9 using the following exposure guideline: for 5µg genomic DNA - probe 33.15/3 hours, probe 33.5/18 hours. Chemiluminescence continues for up to 5 days, therefore re-exposure is possible, but longer exposure times may be required.
  14. Dehybridisation: NICETM probes can be easily removed from membranes by agitating them for 15 minutes in 0.1% SDS at 80 to 85oC. Rinse in 1 x SSC and dry prior to hybridisation or storage.

REAGENTS

1. 0.5M di-sodium hydrogen phosphate buffer pH 7.2:

  • 71g Na2HPO4 made up to 900ml with Milli Ro water
  • Adjust pH to 7.2 with concentrated orthophosphoric acid.
  • Adjust volume to 1L with Milli Ro water.
  • Autoclave
  • Store at room temperature for up to 6 months

2. 10% sodium lauryl sulphate (SDS):

  • 100g SDS
  • Make up to 1L with Milli Ro water
  • Store at room temperature for up to 6 months

3. 20 x standard saline citrate (SSC):

  • 88.2g Trisodium citrate
  • 175.3g NaCl
  • Make up to 900ml with Milli Ro water
  • Adjust pH to 7.0
  • Adjust volume to 1L with Milli Ro water
  • Autoclave
  • Store at room temperature for up to 6 months

4. Prehybridisation buffer:

  • 990ml 0.5M Na2HPO4 pH 7.2 (see 1)
  • 10ml 10% SDS (see 2)

5. Membrane blocking reagent (10% Casein Hammersten):

  • Dissolve 10g Casein Hammersten in 100ml of wash solution 2 (see below) by heating to 50 to 70oC for 1 hour
  • Aliquot (labelled 50ml tubes)
  • Autoclave
  • Store at <-10oC for up to 1 year

6. Hybridisation buffer:

  • Mix 225ml pre-warmed (49-51oC), prehybridisation buffer and 25ml membrane blocking reagent in a clean container for immediate use. Small volumes can be prepared by pro-rata reduction of each component.

WASH SOLUTIONS

Prepare 1 litre of each of solutions 1 and 2 per 2 membranes

  1. Dilute 160ml 0.5M Na2 HPO4 (pH 7.2) to 900ml with Milli Ro water in a clean container and add 10ml 10% SDS. Make up to 1 litre with sterile distilled water and autoclave. Store at room temperature for up to 3 months.
  2. Dissolve 13.8g maleic acid and 8.7g sodium chloride in 900ml Milli Ro water in a clean container.
  3. Adjust pH to 7.5 (care, changes quickly at end point) with concentrated sodium hydroxide (eg. 10M) and make up volume to 1 litre with Milli Ro water.

Autoclave. Store at room temperature for up to 3 months.

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

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