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problems purifying plasmid DNA after restriction digestion


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#1 Raulduke

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Posted 07 March 2005 - 02:28 PM

I've been having problems cleaning up my plasmid DNA following linearization. I've been using qiagen's Qiaquick PCR purification handbook, which I have been told my the manufacturer should work, but I always lose approximately 50% of the DNA following elution with EB. It doesn't matter whether I use 50ul or 30ul and let it sit for 1 minute.

Has anyone else experienced this, or does anyone have any suggestions?

thanks

Raul Duke

#2 methylnick

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Posted 07 March 2005 - 04:50 PM

I always lose approximately 50% of the DNA following elution with EB. 

Has anyone else experienced this, or does anyone have any suggestions?

Hi Raul,

I think with most purification kits, you do lose DNA in the process, most manufacturer's claim to have greater than 90% retention, I seldom get this.

One thing to try is maybe heating your elution buffer to about 50C and then adding this to the column and allow to incubate for at least one minute and then spinning down.

Cheers

Nick

#3 fred_33

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Posted 08 March 2005 - 12:27 AM

hi
methylnick is right. I'm using kits and they recommend to heat the elution buffer to 70░! and stay at least 1' before spin.

if you prefer an old method, and due to the fact it's only a linearization, you can add ddH2O up to 500Ál, treat with proteinase K, and do a phenol/chlo extraction. But columns are better, faster and more convenient obviously!

Cheers,
Fred

#4 Raulduke

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Posted 08 March 2005 - 05:51 AM

Thanks for the help and comments. I get the same information from the company about getting 90% recover, but obviously I've never gotten that percentage recovered. I've heated to 50C, but I'll try 70C next time. Thanks, again




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