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Hi-density screening with Unigene probe

Protocol for hybridization of high density arrayed Bacterial Artificial Chromosome nylon filter blots with 100 PCR isolated Unigene cDNA inserts, pooled in a combined probe

(Steve Chappell Mitchell, California Institute of Technology)

Contents

Isolation and purification of cDNA inserts using polymerase chain reaction

  1. Thaw LB/10% glycerol stock plates containing Unigene clones.
  2. To a 96 well PCR, thin walled plate containing 99µl of sterile ddH2O per well, add 1µl of Unigene culture taken from the bottom of the glycerol stock wells.
  3. Run BOIL program on the MJ Research Inc. thermocycler.
    4. 100°C 5min
  4. Immediately place PCR plate on ice.
  5. Transfer 1µl of boiled culture diluent into the wells of a new 96 well PCR plate.
  6. Aliquot 24µl of PCR reaction mixture per sample well.
    7.          Mixture      (each sample)         2.5µl        10X reaction buffer (Perkin Elmer)         2.5µl        MgCl2 (25mM)         2.0µl        dNTPs (2.5mM)         1.0µl        vector primer 1 (5µM)         1.0µl        vector primer 2 (5µM)         0.5µl        AmpliTaq (Perkin Elmer)        14.5µl        ddH2O
  7. Run PCR thermocycles.
    8.  	Cycles	Time	               No. 	94°	3min	                1 	94°(30sec)55°(30sec)72°(50sec) 30 	72°	5min	                1 	 4°	hold
  8. Stop with 2.8µl loading buffer.
  9. Add 10µl or more of PCR samples to each well of a 1% low melt Sea Plaque GTG agarose, 1XTAE gel. Include a molecular weight marker lane.
  10. Run standard parameters depending on type gel box used.
  11. Cut out PCR product insert bands and place in 1.8ml microcentrifuge tubes.
  12. Incubate 2hrs+ in 0.5ml 1X Gelase buffer (Epicentre Technologies) to equilibrate.
  13. Remove all liquid from microcentrifuge tube, leaving the gel band slice.
  14. Incubate band slice tubes at 70°C, 20min.
  15. Transfer directly into 45°C.
  16. Equilibrate 10min
    17. .
  17. Add 1µl of Gelase (1U/µl) enzyme.
  18. Continue 45°C incubation 2hrs+.
  19. Add 1 volume 3M NaAOc.
  20. Add 2.5 volumes 200 proof Ethanol.
  21. Mix
    22. .
  22. Incubate -20°C, 1hr+.
  23. Microcentrifuge 13,500 rpm, 30min, 4°C.
  24. Decant/aspirate supe.
  25. Wash 1X 0.5ml 70% cold ethanol.
  26. Microcentrifuge 13,500 rpm, 10min, 4°C.
  27. Decant supe.
  28. Air dry pellet.
  29. Resuspend pellet in 20µl TE.

Probe labeling

  1. Combine to a total volume of 30µl, 3µl from each of a group of 10 resuspended insert purifications from above into 1.8ml microfuge tube.
  2. Boiling dH2O bath, 5min.
  3. Immediately place on ice.
  4. Add 3µl Hexanucleotide Mix (Boehringer Mannheim, 1277081).
  5. Add 10µl of dNTPs mix (G, T, and C at 0.5mM each, without A).
  6. Add 5µl 3000Ci/mmol 32P-dATP.
  7. Add 3µl of Labeling Grade Klenow (Boehringer Mannheim, 2U/µl, 1008412).
  8. Incubate A) 25°C, overnight or B) 37°C, 2hrs.
  9. Spin G25 column protocol ( 5 Prime ->3 Prime Inc., 5301-397763).
  10. Scintillation Beta count 1µl of each column eluate to derive DPM (specific activity) estimate.

Pooled probe hybridization to high density filters.

  1. Combine eluate from 10 G25 columns above into one 1.8ml microcentrifuge tube.
  2. Add 150µl Human Placental DNA (Sigma, 10.9mg/ml, D-3287).
  3. Add 250µl Modified Hybe Solution: 
     	Reagent		Final 	NaCl		1M 	Tris pH8.0	0.05M 	EDTA		5mM 	SDS		1% 	Dextran Sulfate	10%
  4. Boil, 8min.
  5. Immediately incubate probe 65°C, 2hrs to preanneal.
  6. Prewet filter blots in dH2O tray.
  7. Roll a maximum of 3 high density filter blots together and place into roller hybridization bottle (Robbins Scientific Inc).
  8. Add 25ml of 65°C preheated Modified Hybe Solution to each roller bottle.
  9. Prehybe filter blots in rotating hybe oven, 65°C, 2hrs.
  10. Add probe volume to the filter blots.
  11. Rotate slowly, 65°C, overnight in hybe oven.

Washing and filming blots

  1. Pour off probe/hybe solution volume into radioactive waste.
  2. Wash with preheated 180ml low stringency wash solution, 65°C, 1hr, fast hybe oven rotation. 
     	Low 	SSC		1X 	SDS		0.1%
  3. Repeat low stringency wash.
  4. Wash with preheated 180ml high stringency wash solution, 65°C, 1hr, fast rotation. 
     	High 	SSC		0.1X 	SDS		0.1%
  5. Repeat high stringency wash.
  6. Place wet filters on exposed sheet of film.
  7. Seal filter blot with plastic wrap covering to keep wet.
  8. Place filter blots down on film (Kodak BioMax MS, 8326886) into X-Omatic Regular (Kodak) cassette with regular screens. Note: you may add special MS screens for increased sensitivity.
  9. Incubate -80°C, overnight.
  10. Leave cassettes at 25°C, 20min.
  11. Develop autorads (see a sample film here).
  12. Address positive clones using lightbox and grid overlay (refer to Research Genetics pattern scheme and plate calculation formula) or by CalTech designed computer program.