Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits User Manual

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SingleShot

™

SYBR

Green Kit

Table 2. Preparation of SingleShot cell lysis master mix for nonadherent cells.

Component

Volume per

Well, µl

Volume for 96-Well

Plate, µl

SingleShot Cell Lysis Buffer

4,608

Proteinase K Solution

DNase Solution

Processing of Nonadherent Cells in a 96-Well PCR Plate

1. Prepare fresh on ice the appropriate volume of SingleShot cell lysis master mix

(Table 2). Mix thoroughly and centrifuge. Use within 2 hr.

2. Count the cells. Transfer appropriate number of cells (10–10

cells per well) to a

96-well PCR plate or tube.

3. Centrifuge at 500–1,000 x g for 5 min. Remove as much of the medium as possible

without disturbing the cell pellet.

4. Wash cells with 125 μl room temperature PBS. Centrifuge at 500–1,000 x g

for 5 min. Carefully remove 120 μl of the supernatant using a pipet, leaving
approximately 5 μl PBS in each well.

5. Add 50 μl of SingleShot cell lysis master mix to each well. Pipet up and down

5 times to ensure complete resuspension of the cell pellet.

6. Incubate for 10 min at room temperature, followed by 5 min at 37°C, and 5 min

at 75°C.

7. The cell lysate can be stored on ice for up to 4 hr, at –20°C for up to 2 months,

or at –80°C for up to 12 months.

8. Go to the Preparation of Reverse Transcription Reactions section.