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Add half the shallot mixture to each bowl, and mix.
4. Add the Worcestershire sauce and lemon juice to taste. 5. Add a generous pinch of the vegetable mixture to each of the opened clams on its half shell.
We added 400 μl of polysome buffer (50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/ml RNasin, 1 mg/ml heparin, 100 μg/ml cyclohexamide, and 1× protease inhibitor mixture) to each sample and disrupted the tissue in 1.5 ml microcentrifuge tubes using pellet pestles (749540-0000; Kimble Chase).
Amplification was carried out in 25 µl of ROX PCR Master Mix (SuperArray) containing 0.2 µM of each primer and 1 µl of the reverse transcription reaction mixture to each well of 96 well cell cycle array plate in a 7700 Real-time PCR system (Applied Biosystems) following the manufacturer's protocol.
Aliquot 25 μL of the microsphere mixture to each well.
Then, we added the mixture to each of the three PrP fibrillar forms.
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The monolayers were then washed with medium containing antibiotics before adding 135 µl of virus-Ab mixtures to each well, in duplicate.
Roll the risotto balls across the seed mixture to coat each ball.
PvuII and an appropriate frequent-cutter enzyme were added to each mixture to inhibit unwanted side reactions.
A 2 % Sodium metabisulphite (0.5 ml) was added to each mixture to deoxygenate the system, mixed thoroughly and sealed with liquid paraffin.
Three hundred μL of 95% ethanol was added to each assay mixture to precipitate the residual RNA.
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