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Acetonitrile (1.5 mL) was added into the mixture, vortexed for 10 min using a sphinix vortexer (M37610-33, Barnstead International, USA), and centrifuged (Biofuge Fresco centrifuge, Heraeus, Germany) for 5 min at 10,000 rpm.
The mixture vortexed for 2 min, and then centrifuged (±600×g) for 3 min.
After cooling, 3 mL of distilled water and 2 mL of hexane were added and the mixture vortexed for 2 min.
The samples were cooled on ice for 5 minutes and spun at max speed in a microcentrifuge for 5 min. The aqueous layer was transferred to a fresh tube, an equivalent volume of phenol chloroform:isoamyl alcohol (25∶24∶1) was added, the mixture vortexed for 4 minutes, and then spun at max speed in a microcentrifuge for 5 minutes.
Matrigel (10% v/v) (BD) was added and the mixture vortexed for 10 s and chilled on ice for 10 minutes.
An equal volume of chloroform:isoamyl alcohol (24:1; v/v) was then added to the mixture and the mixture vortexed for few seconds.
Similar(48)
A sample aliquot of 20 μl was added to each tube and the mixtures vortexed for 10 s.
The mixture was vortexed for thorough mixing and incubated on the cycler (72°C, 10 minutes) to enable 3´-A tail.
The mixture was vortexed for 1 min to obtain a uniform mixture after 10 mL of acetonitrile was added to the mixture.
The mixture was vortexed for few seconds to mix it well.
The mixture was vortexed for 5 10 min to obtain a slurry with additional chloroform being added in the case of mixtures with lower surfactant loading.
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