發布時間：2019-08-10 15:09 原文鏈接： HybridizationofHighDensityArrayedBACNylonFilterBlots

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 ddH₂O 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.

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        ddH2O

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 dH₂O 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 ³²P-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).
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 dH₂O 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.
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.
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.

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HybridizationofHighDensityArrayedBACNylonFilterBlots

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

Probe labeling

Pooled probe hybridization to high density filters.

Washing and filming blots

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