a microplate. Add 4 L of RNase (two units per well; see note
below) into each well containing cells as well as the control wells
without cells. Incubate at room temperature for one hour.
Note: DNase-free RNase should be used at a concentration such that
no more than 10 L contains the full number of units needed to
treat a sample. For example, the RNase Cocktail from Ambion has 500
U/ mL activity, so 4 L per well equals two units per well.
Step 3. Add 100 L of 2X CyQUANT GR dye/ cell-lysis buffer
to each of the microplate wells, including the control wells without
cells.
Step 4. Prepare the DNA standard curve by transferring the previously
prepared DNA serial dilutions into the appropriate wells of the microplate,
as defined in the template editor. The plate should contain two controls:
a digest control prepared following the same steps as the cellular samples,
but without cells (no DNA), and a standard curve control/plate blank
containing only 200 L 1X CyQUANT GR dye/cell-lysis buffer (no
DNA and no RNase).
Step 5. Incubate the samples for 2-5 minutes.
Set up the instrument and software
Step 1. Turn on the microplate reader. Launch the SoftMax Pro
software and use the Assays drop-down menu to locate and open the Basic
endpoint protocol in the Basic protocols folder.
Step 2. Set up the Instrument Settings dialog box as shown in
Table 2.
Step 3. Create a template of the assay showing where standards,
unknowns (cells treated with RNase), and plate blanks will be located
on the microplate. To access the Template Editor, click the Template
button in
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Page: All 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Related biology technology :1.
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