help: clean-up total RNA prep
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:
> 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.
> ctebeau at merle.acns.nwu.edu
Here is a protocol for RNA clean-up on QIAGEN-tip 100
(based on "RNA transcript protocol" in the QIAGEN Protocols
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
Brigitte S. Masone
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