Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 3)

© 2014 Bio-Rad Laboratories, Inc. 10042473
SingleShot ™ Probes Kit
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 5 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.
Preparation of Reverse Transcription Reactions
1. For optimal results, reactions should be assembled on ice. Prepare the reverse
transcription reaction according to the directions in Table 3. Mix thoroughly by
pipetting up and down several times.
Table 2. Preparation of SingleShot cell lysis master mix for nonadherent cells.
Table 3. Preparation of reverse transcription reaction.
Component
Volume per
Well, µl
Volume for 96
Reactions, µl
SingleShot Cell Lysis Buffer 48 4,608
Proteinase K Solution 1 96
DNase Solution 1 96
Component Volume per Reaction, µl
5x iScript Advanced Reaction Mix 4
iScript Advanced Reverse Transcriptase 1
Cell Lysate 4–9
Nuclease-Free Water Variable
Total volume 20