8 replies to this topic

[jehane]

Hello All
i have used salting out method for extraction of Genomic DNA from urinary bladder of mice but as the tissue contain large amounts of fats.
so the Extracted DNA is contaminated with lipid.

HOW could i get rid of those fats from my DNA sample???

Edited by jehane, 30 August 2010 - 11:43 AM.

[phage434]

Lipids are very soluble in chloroform, so a simple chloroform extraction will remove them.

[perneseblue]

phage434, on 30 August 2010 - 06:11 PM, said:

Lipids are very soluble in chloroform, so a simple chloroform extraction will remove them.

I agree. That would solve the problem

[jehane]

phage434, on 30 August 2010 - 06:11 PM, said:

Lipids are very soluble in chloroform, so a simple chloroform extraction will remove them.

DNA now in TE buffer how could i apply chloroform extraction on it???

[bob1]

The same as you would for any other chloroform extraction - add 1 volume of chloroform to the sample, shake or vortex vigourously for 30 s, then centrifuge to separate layers...

[jehane]

bob1, on 02 September 2010 - 03:56 PM, said:

The same as you would for any other chloroform extraction - add 1 volume of chloroform to the sample, shake or vortex vigourously for 30 s, then centrifuge to separate layers...

without need for phenol:chloroform step at first?

[perneseblue]

jehane, on 02 September 2010 - 04:04 PM, said:

bob1, on 02 September 2010 - 03:56 PM, said:

The same as you would for any other chloroform extraction - add 1 volume of chloroform to the sample, shake or vortex vigourously for 30 s, then centrifuge to separate layers...

without need for phenol:chloroform step at first?

Yes, just add chloroform.

The purpose of phenol is to denature proteins. Since proteins aren't a problem here, there is no need for phenol:chloroform mixture.

However if you don't have chloroform and only have phenol-chloroform, you can use phenol-chloroform mixture instead.

[phage434]

I'd recommend an ethanol precipitation and resuspension in TE following the chloroform extraction.

[DNAgeek]

Also, there are kits for extraction of DNA or RNA from high-lipid tissues. I use the RNeasy Lipid Tissue Mini Kit for RNA, but there should be some for DNA as well.