RNA Purification via Spin column kit

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dinofernando
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Joined: Sat Aug 04, 2007 1:50 am
Location: Oshawa, Canada UOIT

RNA Purification via Spin column kit

Post by dinofernando » Thu Mar 17, 2011 6:47 pm

Hi everyone,

I'm having trouble with extracting RNA via Qiagen spin column kit. Every time I do the procedure I get excellent yields around 1000 - 2500 ng/uL but horribly low purity 1.5 - 1.7 A260/A280. I know for pure RNA I need a ratio of ~ 2.0. I'm going nuts people.

I'm growing my cells to OD of 0.8 - 1.0 and pelleting, snap freezing and storing in -80C for a few days.

Can anyone suggest at what step I'm getting all this contamination? I find the longer I mix with Lysozme and then buffer RLT + B-mercapto the lower my concentration of RNA.

I'm going to go try it again and reduce my RLT-beta mercapto incubation time.

Any suggestions you can afford would be great thanks guys.

-Dinesh.
-Dinesh
Ph.D. Student
UOIT

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