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Amplification primers and reaction buffer were removed from each sample using the AMPure Kit (Agencourt, Danvers, MA).
Traces of buffer were removed from the pellet and it was resuspended in 25 μl of protein loading buffer, heated to 95°C for 8 min, and gently centrifuged for 30 sec at 2000 × g.
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At predetermining time points, hydrogels were collected with a magnetic separator and excess buffer was removed from the tubes.
After this, all excess Krebs buffer was removed from the well leaving only the pinned out tissue.
After 2 h hybridization at 46°C, the hybridization buffer was removed from the samples.
Following the final wash, all buffer was removed from the beads and 20 µl of 2X sample buffer was added to the beads.
The wash buffer was removed from the filter as above, the glass syringe filter with the bound DNA attached to a new 5 ml syringe and 1 ml of TE (pH 8.0) was added.
Next, the filters were washed with 20 ml of wash buffer (2 mM Tris.HCl pH 7.5, 20 mM NaCl in 80% EtOH), and then the wash buffer was removed from the filters by repeatedly pressing air through the glass syringe filter.
RNAlater buffer was removed from dissected midguts and the tissues homogenized with 27G syringes in TRIzol reagent (Ambion, Life Technologies).
The Offgel IEF buffer was removed from all the wells, and 150 µl of peptide sample prepared in Offgel IEF buffer was added to each well.
The remaining small amount of buffer was removed from the tube and the slices were weighted to obtain the wet tissue weight.
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