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Low OD using Trizol to isolate total RNA from cell lines

Low OD using Trizol to isolate total RNA from cell lines - Protocols and Methods Forum

Low OD using Trizol to isolate total RNA from cell lines - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-03-2004, 04:42 AM
Alex
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Default Low OD using Trizol to isolate total RNA from cell lines



Hi all,

I use Trizol to isolate total RNA from mouse cell line. Sometimes I
get a very poor 260/280 ratio (0.9-1.2). I think it might be due to
protein contamination. Has anyone there ever tried any modification to
this protocol to get a good yield and better 260/280 ratio? What
method do you use to purify the total RNA after you check with a
spectrophotometer( besides phenol:chloroform extraction from Maniatis
)?

Thanks,

Alex
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  #2  
Old 05-03-2004, 08:20 AM
Peter Ellis
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Default Low OD using Trizol to isolate total RNA from cell lines

[Only registered users see links. ] wrote:

We use TRI reagent (Sigma) - equivalent to Trizol, followed by an
RNEasy cleanup (QIAGEN). The yield is lower since you lose low weight
species such as tRNA, but if you're looking at mRNAs that makes no
difference. If you want to use it for RT-PCR, you'll probably still
need to do a DNAse digest, or simply design primers so they won't
amplify anything from genomic DNA.

Peter
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  #3  
Old 05-03-2004, 01:43 PM
Sell Biology
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Default Low OD using Trizol to isolate total RNA from cell lines

[Only registered users see links. ] (Alex) wrote:


Don't some protocols use a proteinase K digestion, perhaps at a lower
temperature for a longer time? Just in case the purity is not where they
like it. Make sure your pH is not above neutral.

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  #4  
Old 05-16-2004, 07:02 AM
brenda
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Default Low OD using Trizol to isolate total RNA from cell lines

Hi Alex,

I use Tripure (Roche Diagnostics) for many years now and I have never seen
this effect. Perhaps you should use another manufacturere.

Brenda

"Alex" <[Only registered users see links. ]> schreef in bericht
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