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RNA extraction troubleshooting: low 260/230


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#1 ginger88

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Posted 03 March 2011 - 02:41 PM

I've been working with RNA in Trizol for quite a while for use in microarrays, and by and large, most of my samples turn out fine. But I need to boost the 260/230 ratio for a significant number of samples, and I have been completely stonewalled. I've tried the "barrel-roll" method (rolling the Qiagen spin column during the wash steps to really wash out all the RNA) recommended elsewhere, which seems to work some of the time. But I need something that works consistently. I've also read about using a combination of 1-butanol and water saturated diethyl ether, where the butanol is supposed to clean up the phenol contamination that's apparently causing the low 260/230, and the ether cleans off the butanol. I've been playing around with a protocol developed from the Krebs et al 2009 paper, and again, I've found some success, but not complete success. Part of what concerns me is that in using the butanol/ether combo for a couple of times as recommended by this paper is that my aqueous phase gradually turns into a pellet, though the paper describes it as liquid phases. After getting this pellet, I've been resuspending the pellet in DEPC water and using a purification protocol involving isopropanol and high salt solutions before purifying in the spin column. At best, this method works about half the time. Has anyone had any consistent success in using this butanol/ether step, and if so, how? Or has anyone else found any other useful methods for removing phenol and other aromatic contamination?

#2 pDNA

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Posted 04 March 2011 - 08:01 AM

You ever heard of safe-lock tubes? ...they really work fine!

they really can boost your purity! ...you can check this page, it has quite a lot information on that topic!

Good luck!

Regards,
p

I've been working with RNA in Trizol for quite a while for use in microarrays, and by and large, most of my samples turn out fine. But I need to boost the 260/230 ratio for a significant number of samples, and I have been completely stonewalled. I've tried the "barrel-roll" method (rolling the Qiagen spin column during the wash steps to really wash out all the RNA) recommended elsewhere, which seems to work some of the time. But I need something that works consistently. I've also read about using a combination of 1-butanol and water saturated diethyl ether, where the butanol is supposed to clean up the phenol contamination that's apparently causing the low 260/230, and the ether cleans off the butanol. I've been playing around with a protocol developed from the Krebs et al 2009 paper, and again, I've found some success, but not complete success. Part of what concerns me is that in using the butanol/ether combo for a couple of times as recommended by this paper is that my aqueous phase gradually turns into a pellet, though the paper describes it as liquid phases. After getting this pellet, I've been resuspending the pellet in DEPC water and using a purification protocol involving isopropanol and high salt solutions before purifying in the spin column. At best, this method works about half the time. Has anyone had any consistent success in using this butanol/ether step, and if so, how? Or has anyone else found any other useful methods for removing phenol and other aromatic contamination?






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