( ...)Easy preparation of total RNA from cultured cells with TRIzol and ,,, Eppendorf Phase Lock Gel

Christian Bauer, Oliver Klotzsche, and Frank Apostel
Christian Bauer, Oliver Klotzsche, CCS Cell Culture Service GmbH, Falkenried 88, D-20251 Hamburg, Germany,
Frank Apostel, Eppendorf AG, Germany Introduction

The classic RNA isolation procedures are based on a mono-phasic solution of phenol and guanidinium isothiocyanate solutions, followed by organic extraction and alcohol precipitation of the RNA.1 TRIzol is the commercial version of this popular single-step method of total RNA isolation. This highly reliable technique performs well with both small and large quantities of tissues or cultured cells and allows simultaneous processing of a large number of samples. The Eppendorf Phase Lock Gel (PLG) separates the aqueous and organic phase with a solid barrier and eases handling. PLG is inert, heat stable and compatible with enzymatic reactions of nucleic acids. The procedure can decrease processing time and improve nucleic acid recovery from organic extraction purifications, all while minimizing user exposure to hazardous organic solutions.2

In this report we describe the use of Eppendorf Phase Lock Gel (PLG) Heavy for the isolation of total RNA from human tissue culture cells and present data regarding the quality, quantity and utility of the RNA recovered.

Materials and Methods

Isolation of total RNA from Jurkat cells

The following protocol was performed to isolate total RNA from cultured eukaryotic cells. The organic extraction of one half of the samples was processed in pre-spun (2030 seconds at 12,000 x g) 2 ml PLG Heavy tubes. The other half was processed in 2 ml standar
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