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1 year ago

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

Experiment:

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The first thing you can try is to attempt phase separation in room temperature instead of 4°C. After using phenolic compounds and chloroform the RNAses won’t be active so there is no risk of degradation. After adding the chloroform you can also let the samples sit for 10 min at RT, vortex them and finally centrifuge them at around 4000-6000 rpm. You can also add isoamyalkohol (IAA) to the chloroform in a ratio of 1:24 (IAA/chloroform) which might improve the success rate for separation.

Answered 1 year ago


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you should see two layers - an upper chloroform layer and an aqueous lower layer - assume precipitated material in the  middle.  With lysis/chloroform addition, you certainly should have seen both layers. Suppose it's possible that excessive mixing  might have effected an emulsion but hard to see that as stable. Suggest you call the Qiagen folks for help.

Answered 1 year ago


The first thing you can try is to attempt phase separation in room temperature instead of 4°C. After using phenolic compounds and chloroform the RNAses won’t be active so there is no risk of degradation. After adding the chloroform you can also let the samples sit for 10 min at RT, vortex them and finally centrifuge them at around 4000-6000 rpm. You can also add isoamyalkohol (IAA) to the chloroform in a ratio of 1:24 (IAA/chloroform) which might improve the success rate for separation.

Answered 1 year ago


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