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RNeasy Mini Handbook 10/2019
31
2. Disrupt the cells by adding Buffer RLT.
For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add the
appropriate volume of Buffer RLT (see Table 5). Vortex or pipet to mix, and proceed to
step 3.
Note: Incomplete loosening of the cell pellet may lead to inefficient lysis and reduced
RNA yields.
Table 5. Volumes of Buffer RLT for lysing pelleted cells
For direct lysis of cells grown in a monolayer, add the appropriate volume of Buffer RLT (see
Table 6) to the cell-culture dish. Collect the lysate with a rubber policeman. Pipet the lysate
into a microcentrifuge tube (not supplied). Vortex or pipet to mix, and ensure that no cell
clumps are visible before proceeding to step 3.
Table 6. Volumes of Buffer RLT for direct cell lysis
* Regardless of the cell number, use the buffer volumes indicated to completely cover the surface of the dish.
3. Homogenize the lysate according to step 3a, 3b, or 3c.
See “Disrupting and homogenizing starting material”, page 22, for more details on
homogenization. If processing ≤1 x 10
5
cells, homogenize by vortexing for 1 min. After
homogenization, proceed to step 4.
Number of pelleted cells Volume of Buffer RLT (μl)
<5 x 10
6
350
5 x 10
6
– 1 x 10
7
600
Dish diameter (cm) Volume of Buffer RLT (µl)*
<6 350
6–10 600
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