4 replies to this topic

[LeeBrook]

Hi,

I've been using a protocol to extract DNA from blood parasite in whole blood using a lysis buffer with TrisHCl, EDTA, SDS and Triton X-100, and a proteinaseK step.

The lysed material is then treated with a phenol:chloroform extraction and when spun I am getting what I can best describe is like a layer of soap between the organic and very tiny aqueous phase.

 

This protocol had been working just fine and suddenly all samples get this "soap" layer.

 

I have remade solutions, obtained new proteinase K and new phe:chl.

In addition I have pH'd everything and nothing appears abnormal.

 

any suggestions would be gratefully received!!

thanks

Lee

[mdfenko]

are you sure the layer is soap? it may be lipid. blood has variable amounts of lipid. maybe this time the blood samples are high in lipid.

 

it shouldn't be a problem if you can still isolate the phases.

[LeeBrook]

I think it maybe lipids - but I thought those would have been lost in the phenol:chloroform?!?  I'm rather confused And its about 30% of the total volume and a white-ish/brown-ish blob between the organic and aqueous layers?!?

Any ideas are gratefully received!!

thanks

[phage434]

This layer is common and expected in phenol chloroform extractions. It is mostly the proteins of your sample, which are denatured. They typically have both hydrophobic and hydrophilic regions of the molecule, and segregate at the boundary. If there is too much of this layer, you can increase the volume of the sample by dilution and the volume of the phenol-chloroform. You lose sample in any extraction such as this, but there is little to be done about it. You want to avoid this layer when picking up the water layer. Depending on your next step, a second extraction with phenol chloroform, or with chloroform alone will help. The chloroform alone extraction will remove most of the phenol, which can be important if your next step is inhibited by its presence. The layer is not lipids -- they will fully dissolve in the phenol chloroform or chloroform layers, but they could be lipoproteins. In any case, you don't want them.

[LeeBrook]

Thanks so much!!

I'll try increasing my volumes.

 

Do you think a longer proteinase K step would be good?

I'm currently running 30min at 50oC