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文献和实验Replication timing by density transfer
), arrest with 200 nM alpha factor (1-1.25 doubling times, until % unbudded cells stops increasing). NOTE: This alpha factor concentration is for bar1 strains. BAR1+ strains require 15x more alpha factor. 3. Filter, wash in Ycomp+alpha factor, resuspend
Gene Knockout Techniques(1) The Knockout Process
to a single well of a 12-well untreated tissue culture plate (Evergreen #222-8046-01F).Add 6.7 ul TMP working stock (0.3 mg TMP/ml acetone) to each 1 ml suspension of worms (final concentration 2 ug/ml). It?s best to do this in a fume hood, probably under dim
in methanol at room temp. 3. Remove methanol and add 1x Blotting buffer until ready to use. G. Membrane transfer 1. Assemble "sandwich" for Bio-Rad's Transblot. 2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer
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