Hello,

 

I have phase separation problems after homogenization and chloroform addition. Some of the samples have a very thick interphase and little/no aqueous phase after centrifugation and once inverted aqueous phase. What will be the cause of this situation and how should I overcome this?

 

If you shake a chlorinated solvent with water to vigourously, then you often get an emulsion that takes a VERY long time to seperate. The best way to resolve the issue is to leave it for several hours, or overnight, and the two layers will eventually seperate. If you can't wait that long, then you could try sonicating the mixture, it might help them seperate. However, sonicating the mixture will heat it up which may cause your RNA to degrade.

Thank you for the response but I still am confused as I didn't add water to the sample!

Here is the procedure I followed,

 

1. Lysed and homoginized the tissue sample with 1 ml of qiazol

 

2. spun the sample through qia shredder tube, 1000 RPM, 1 min, @ RT

 

3. added 200 micro litters of chloroform

 

4. vortex well

 

5. spun at 9400 RPM, 15 min, @4'C

 

And after the 5th step I am supposed to see 3 phases of RNA, DNA and Protein....And I did not see any RNA (Aqueous phase!)...So can you tell me where did I do anything wrong in this 5 step protocol??

 

Also I have another question regarding RNA quality analysis,

 

I collected some RNA and quantified it using nanodrop, concentrations are good and 260/280 ratios are also good : between 1.8 and 2

 

But when I ran agarose gel(1%, with 5 micro liter of ethidium bromide, Samples: 1 micro gram of samples with 6X loading buffer ), all the samples gave smear.....no bands!

 

Do you have any idea why did that happen??

Unfortunately, while techincally I'm a chemical biologist, I havn't got around to doing the biology of my PhD yet so in terms of why it doesn't work Im not sure...I'll ask the biologists in my group if they know whats going on.

 

You said in your original post that you could not see the aqueous phase after you had mixed it with the chloroform? So there is already water there...

Alriht..let me know if you get any further information regarding the issue,

 

Thank you for the helpful responses

But when I ran agarose gel(1%, with 5 micro liter of ethidium bromide, Samples: 1 micro gram of samples with 6X loading buffer ), all the samples gave smear.....no bands!

Strong smears are often due to DNA contamination, or serious degradation. Some smear is expected, though the rRNA bands should be the strongest.

 

The water comes from your lysis buffer and the sample itself. If no phase separation is observed, it is often one of the following :

 

- contamination of sample or reagents with organic solvent. If the chloroform is e.g. contaminated with isoamylalcohol there will be a no phase separation

- incomplete mixing of the homogenate: mix again, let rest at RT for a few mins then centrifuge

Hi CharonY,

 

Thank you for the response. It helped me to understand the procedure more clearly! but I wonder what is the importance of letting the samples sit at RT for a while and then centrifuge??

Edited January 17, 201114 yr by search

It gives time for the homogenate to settle a bit and allow for more thorough precipitation.

Alright! Thank you so much..