3 replies to this topic

[Hannah_be]

I have a sample containing more than 250 µg of DNA fragments between 4 - 10kb. This DNA has been extracted by phenol:chloroform and ethanol precipitation. The 260/280 ratio is however very high (5) probably due to phenol contamination. Now I wondered if there are ways to purify this sample. Commercial purification techniques like the kits from qiagen (MinElute Reaction Cleanup Kit  or QIAquick PCR Purification Kit) can only purify up to 10µg so that is not an option. Does anyone have suggestions?

[microgirl]

You won't like this answer, but can you divide it into 10 ug aliquots and then run them individually thru the commercial clean up kits? You'll probably have to ethanol precipitate when you're done though.

Edited by microgirl, 27 October 2009 - 09:38 AM.

[phage434]

A glassmilk or QiaexII binding of the DNA would work well.  You could also use columns intended for maxiprep to bind and elute the DNA.

But you could probably get rid of phenol with simple chloroform extractions followed by ethanol precipitation.

[Prep!]

Hannah_be, on Oct 27 2009, 07:31 PM, said:

I have a sample containing more than 250 µg of DNA fragments between 4 - 10kb. This DNA has been extracted by phenol:chloroform and ethanol precipitation. The 260/280 ratio is however very high (5) probably due to phenol contamination. Now I wondered if there are ways to purify this sample. Commercial purification techniques like the kits from qiagen (MinElute Reaction Cleanup Kit  or QIAquick PCR Purification Kit) can only purify up to 10µg so that is not an option. Does anyone have suggestions?

Cant you dilute and do different preps (i know its tedious!!!)
or alternatively you can use a small scale Q column for purification. Even the samples will be better concentrated along with pure DNA!!!
Best luck!