Serum/plasma kit

[Fanatic_scientist]

Good morgning,

I used a serum/plasma kit. After phase separation there should be 3 phases. Aqueous phase, white interphase and a red organic phase. In some of samples there did not create an aqueous phase after some time. Does anyone know the solution to this problem? Thanks in advance!

[PhilGeis]

Can you provide a link to the "kit"?  This sounds like you've just spun down a tube of blood.  Plasma, serum?  Was an anticoagulant used?

 

[Fanatic_scientist]

Sorry for the late reply,

Thanks for your answer.

I used this kit:

- https://www.labettor.com/combos/detail/id/3413/  

-https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/mirna/mirneasy-serumplasma-kit/#productdetails. 

 

I used it for miRNA purification in Plasma from human blood samples. In the end I will use it for qPCR. 

[PhilGeis]

I think  I understand but don;t think I can help..  From 2nd link, 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.

[Fanatic_scientist]

Okay, I will ask Qiagen as well then! 

Thanks for your answers;)

[CharonY]

Looks like typical choloroform/phenol extraction protocol. I have not experienced myself but in general there a couple of theoretical effects that one might need to control to make sure that good phase separation happens. This includes having enough ionic strength of the aqueous layer, control of pH or sometimes even just the addition of another organic solvent. I suspect that bad phase separation can happen due to excess amounts of compounds that in a given poorly separate into either phase, which includes certain proteins and lipids.