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Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents

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Important: Lysis reagent is formulated as a 2X solution. The final lysis buffer concentration in any lysis step should always be 1X for maximum RNA recovery.

Method 1 Automated lysis reagent addition by the 6700 workstation

1.Pellet suspension cells by centrifugation.

2.Remove the growth media by aspiration from all wells, taking care to ensure that the cell pellet is not disturbed.

3.Re-suspend cell pellet in 125 μL PBS (phosphate buffered saline, Ca2+/Mg2+ free).

4.Place the cell culture plate containing the cells in the primary input position of the 6700 workstation.

5.Activate the default lysis protocol (Figure 2).

6.Configure the lysis protocol to add 125 μL of 2X lysis reagent and to mix the sample at least 3 times after addition.

7.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

8.If the default RNA archive protocol is used, purified RNA will be eluted in 150 μL of PCR compatible buffer.

Method 2 Manual lysis of suspension cells

1.Pellet suspension cells by centrifugation.

2.Remove the growth media by aspiration from all wells, taking care to ensure that the cell pellet is not disturbed.

3.Re-suspend cell pellet in 250 μL of a 1:1 mixture of lysis reagent and PBS (phosphate buffered saline, Ca2+/Mg2+ free).

4.Ensure complete nucleic acid release by taking up the lysate into the pipette tip at least three times, mixing the lysis buffer with the cell pellet.

5.Place the cell culture plate containing the lysate in the primary input position of the 6700 workstation.

6.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

7.If the default RNA archive protocol is used, puri
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