help: clean-up total RNA prep

QIAGEN qiagen at kaiwan.com
Fri Oct 13 13:25:41 EST 1995


In article <45hhhb$grl at news.acns.nwu.edu>, ctebeau at melre.acns.nwu.edu
(Christopher M. Tebeau) wrote:

> Hello,
> 
>   I've recently isolated total RNA from cilliated protozoans using GTC 
> and cscl spinning then pc extractions, but the 260/280 are still showing 
> contamination.  Rather than more extractions, would it be possible to 
> clean these on a Qiagen tip-100 (used for plasmid preps).  I have these 
> and I've heard it can be done but I have no specifics.  Any help would be 
> greatly appreciated.
>                                                 Thanx,
>                                                 Chris
>                                                 
> ctebeau at merle.acns.nwu.edu


Chris,

Here is a protocol for RNA clean-up on QIAGEN-tip 100 
(based on "RNA transcript protocol" in the QIAGEN Protocols
Handbook/Summer 1992):

1.    Adjust the volume of sample to 10 ml by adding Buffer QA.
Note: RNA sample should not contain any anionic detergents such as SDS.
The final salt concentration of the sample plus Buffer QA should be no
more than 400 mM.
2.    Equilibrate a QIAGEN- tip 100 with 3 ml Buffer QAT.
3.    Apply the sample from step 1 onto the QIAGEN-tip and allow it to
enter the resin by gravity flow.
4.    Wash the QIAGEN-tip with 12 ml Buffer QA.
5.    Elute the RNA with 6 ml of Buffer QRU into a clean tube.
Note: Prewarming Buffer QRU to 45°C imporves elution.
6.    Precipitate the RNA with one volume of ice-cold isopropanol, mix and
icubate on ice for 10 min. Centrifuge 15,000xg for 30 min at 4°C.
7.    Wash RNA pellet with 70% ethanol, air dry for 10 min, and redissolve
it in a suitable volume of buffer.

Buffer QA:   400 mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0
Buffer QAT:  400 mM NaCl, 50 mM MOPS, 15% ethanol, 0.15% Triton X-100, pH 7.0
Buffer QRU:    900 mM NaCl, 50 mM MOPS, 15% ethanol, 6 M urea, pH 7.0 ‹
should be made fresh before use. 

Alternatively, a Buffer QR stock solution can be made and urea added just 
prior to use.
Buffer QR: 1.2 M NaCl, 67 mM MOPS, 20% ethanol, pH 6.7 (3.62 g of urea added 
to 7.5 ml of Buffer QR will give 10 ml of ready-to-use QRU buffer)

If you have any questions, please give QIAGEN Tech Service a call at
800-426-8157.

Brigitte S. Masone
Product Manager



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