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

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SingleShot

™

SYBR

Green Kit

Table 1. Preparation of SingleShot cell lysis master mix for adherent 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 Adherent Cells in a 96-well Culture Plate

For processing adherent cells in non–96-well cell culture plates, refer to Table 1

in Appendix A

For processing trypsinized adherent cells, neutralize the trypsin with culture

medium. Follow instructions in Processing Nonadherent Cells in a 96-Well
PCR Plate section

1. Seed the cell culture in advance in a 96-well culture plate so that the cell numbers

at harvest are in the range of 100,000–10 cells/well.

For adherent cells, it is important to use cells that are fully adhered to the plate

to avoid cell loss during washing

Using too many cells may result in incomplete cell lysis and can inhibit RT-qPCR.

For optimal results, we recommend using the SingleShot RNA control included
in this kit to determine the appropriate input cell number

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

(see Table 1). Mix thoroughly and centrifuge. Use within 2 hr.

3. Remove cell culture medium completely by aspiration.
4. Wash cells with 125 μl of room temperature PBS. Aspirate to remove

PBS completely.

5. Add 50 μl of SingleShot cell lysis master mix to each well.
6. Incubate without agitation for 10 min at room temperature.

Do not mix the cells with the solution by pipetting. For step 6, do not exceed

20 min at room temperature

7. Transfer the cell lysate to a PCR plate or centrifuge tube. Incubate at 37°C for

5 min, followed by 5 min at 75°C.

Use a thermal cycler for best thermal uniformity

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

or for up to 12 months at –80°C.

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