Hybridization of High Density Arrayed BAC Nylon Filter Blots
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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
Isolation and purification of cDNA inserts using polymerase chain reaction
- Thaw LB/10% glycerol stock plates containing Unigene clones.
- To a 96 well PCR, thin walled plate containing 99µl of sterile ddH2 O per well, add 1µl of Unigene culture taken from the bottom of the glycerol stock wells.
- Run BOIL program on the MJ Research Inc. thermocycler. 100°C 5min
- Immediately place PCR plate on ice.
- Transfer 1µl of boiled culture diluent into the wells of a new 96 well PCR plate.
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Aliquot 24µl of PCR reaction mixture per sample well.
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 ddH2 O -
Run PCR thermocycles.
Cycles Time No. 94° 3min 1 94°(30sec)55°(30sec)72°(50sec) 30 72° 5min 1 4° hold
- Stop with 2.8µl loading buffer.
- 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.
- Run standard parameters depending on type gel box used.
- Cut out PCR product insert bands and place in 1.8ml microcentrifuge tubes.
- Incubate 2hrs+ in 0.5ml 1X Gelase buffer (Epicentre Technologies) to equilibrate.
- Remove all liquid from microcentrifuge tube, leaving the gel band slice.
- Incubate band slice tubes at 70°C, 20min.
- Transfer directly into 45°C.
- Equilibrate 10min.
- Add 1µl of Gelase (1U/µl) enzyme.
- Continue 45°C incubation 2hrs+.
- Add 1 volume 3M NaAOc.
- Add 2.5 volumes 200 proof Ethanol.
- Mix.
- Incubate -20°C, 1hr+.
- Microcentrifuge 13,500 rpm, 30min, 4°C.
- Decant/aspirate supe.
- Wash 1X 0.5ml 70% cold ethanol.
- Microcentrifuge 13,500 rpm, 10min, 4°C.
- Decant supe.
- Air dry pellet.
- Resuspend pellet in 20µl TE.
Probe labeling
- 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.
- Boiling dH2 O bath, 5min.
- Immediately place on ice.
- Add 3µl Hexanucleotide Mix (Boehringer Mannheim, 1277081).
- Add 10µl of dNTPs mix (G, T, and C at 0.5mM each, without A).
- Add 5µl 3000Ci/mmol 32 P-dATP.
- Add 3µl of Labeling Grade Klenow (Boehringer Mannheim, 2U/µl, 1008412).
- Incubate A) 25°C, overnight or B) 37°C, 2hrs.
- Spin G25 column protocol ( 5 Prime ->3 Prime Inc., 5301-397763).
- Scintillation Beta count 1µl of each column eluate to derive DPM (specific activity) estimate.
Pooled probe hybridization to high density filters.
- Combine eluate from 10 G25 columns above into one 1.8ml microcentrifuge tube.
- Add 150µl Human Placental DNA (Sigma, 10.9mg/ml, D-3287).
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Add 250µl Modified Hybe Solution:
Reagent Final NaCl 1M Tris pH8.0 0.05M EDTA 5mM SDS 1% Dextran Sulfate 10%
- Boil, 8min.
- Immediately incubate probe 65°C, 2hrs to preanneal.
- Prewet filter blots in dH2 O tray.
- Roll a maximum of 3 high density filter blots together and place into roller hybridization bottle (Robbins Scientific Inc).
- Add 25ml of 65°C preheated Modified Hybe Solution to each roller bottle.
- Prehybe filter blots in rotating hybe oven, 65°C, 2hrs.
- Add probe volume to the filter blots.
- Rotate slowly, 65°C, overnight in hybe oven.
Washing and filming blots
- Pour off probe/hybe solution volume into radioactive waste.
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Wash with preheated 180ml low stringency wash solution, 65°C, 1hr, fast hybe oven rotation.
Low SSC 1X SDS 0.1%
- Repeat low stringency wash.
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Wash with preheated 180ml high stringency wash solution, 65°C, 1hr, fast rotation.
High SSC 0.1X SDS 0.1%
- Repeat high stringency wash.
- Place wet filters on exposed sheet of film.
- Seal filter blot with plastic wrap covering to keep wet.
- 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.
- Incubate -80°C, overnight.
- Leave cassettes at 25°C, 20min.
- Develop autorads (see a sample film here ).
- Address positive clones using lightbox and grid overlay (refer to Research Genetics pattern scheme and plate calculation formula) or by CalTech designed computer program.
