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Trouble in siRNA extraction


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#1 xiangliu

xiangliu

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Posted 05 July 2004 - 06:56 PM

:)
Recently I extracted the 25nt RNA from PTGS tobacco leaves followed the protocol "Extraction of 25NT RNA" provoided by Dr. Hamilton. But I met some troubles during the extraction.

While I used the DEAE-sepharose CL-6b (Amersham Biosciences) to purify the small RNA, I found that most of RNAs existed in the filtrate and the wash buffer (buffer A) and nothing in the elute buffer (buffer B). I tried this with 25nt oligo primer and got the same result .

Posted Image.

Could anybody gave me some advice about this?

The following is the protocol copied form Dr. Hamilton's protocol,

3. I've recently started to use DEAE-sepharose CL-6b (Sigma) in preference to Qiagen columns since it is cheaper. Equilibrate the sepharose in buffer A (50mM MOPS/NaOH pH7; 15% isopropanol; 0.2M NaCl; 0.15% Triton x-100) I make quick columns with this by cutting some whatman 1 filter paper circles small enough to plug the end of a 5ml disposable syringe, adding 1ml of the equilibrated sepharose and letting the column settle. Redissolve the ethanol pptıd low mol. wt. fraction in buffer A and apply to column under force of gravity only. Or..you can skip a step by adding water to the supernatant of the PEG pptn (section 2 above) until the NaCl concentration reaches 0.2M and then adding 5-10 volumes of bufferA and applying this to the column. Collect the flow-through and reapply to column. Wash the column with more bufferA (I do 10 column volumes) and elute with buffer B (same as bufferA except NaCl concentration is 1.0 M). Add three vol of ethanol directly to this eluate and place at -20 for at least two hours and after a 10000xg spin for 20 mins and a 70% ethanol wash, I dry the pellets and redissolve them in formamide (Usually 100-1000ul).

Edited by xiangliu, 05 July 2004 - 06:57 PM.





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