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

erformed with the Eppendorf MasterTaq Kit (50 mM KCl; 10 mM Tris-HCl pH 8.3; 15 mM Mg-Acetate; 1 U Taq DNA Polymerase) and 0.5 M of specific 5 and 3-primers according to the following protocol: The fragments were separated by gel electrophoresis in 1% agarose gel. Forward primer:
5-ATGGAGGAGCCGCAGTCA3
Reverse primer:
5-CGGTCTCTCCCAGGACAG-3

The PCR reaction was performed on the Eppendorf Mastercycler gradient** using the following cycling conditions:

94C 10 minute initial denaturation
35 cycles:
94C 30 seconds
56C 30 seconds
72C 1 minute
72C 5 minute final extension

Results and Discussion

Determination of yield and quality of the isolated RNA

As shown in Fig.1, the isolation of total RNA using Eppendorf Phase Lock Gel results in greater yields compared to conventional organic extraction preparations. The purity of the prepared RNA is better than without PLG when evaluated with absorbance ratios at A260/280.

Fig. 1: Determination of yield and purity of the isolated total RNA by UV spectrophotometry (Eppendorf BioPhotometer). The average yield of total RNA in g and the average of the ratio A260/280 are shown, comparing the samples purified with PLG and without PLG. Denaturing agarose gel electrophoresis

The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). Intact t
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