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Plasmid DNA cleaning


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#1 nbj

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Posted 01 February 2005 - 12:52 PM

Hi all,
i changed amino acids for two of my mutants using site directed mutagenesis, everything worked fine till sequencing started, even the commercial primers could not sequence beyond 200-300 bp, with some samples not giving any results, so could someone suggest simple n easy method to clean DNA after purifiaction using promega miniprep kit, cause the sequencing lab people cleaned and it worked.
thanks
nbj

#2 ros

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Posted 01 February 2005 - 01:59 PM

I think the most efficient way of cleaning your DNA would be a phenol:chloroform:isoamyl alcohol purification and then a sodium acetate/ethanol precipitation.

Normally, miniprep kits come with an elution buffer that contains Tris and EDTA. These buffers can interfere with the sequencing reactions, and so I always elute in purified water prior to sequencing. I would recommed that you try this first, and if it doesnt work, then do the phenol chlorofom purification and precipitation.

Are you eluting in water or elution buffer?

#3 fred_33

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Posted 02 February 2005 - 04:48 AM

hi
i'm using promega miniprep kit and have observed two paricularities :

First it is very important to let your miniprep column air dry correctly and room temperature. At least thirty minutes air-drying are neccessary.

The second one is for the sequencing. I've observed that when i start qith miniprep concentration under 600ng/Ál sequencing was bad . I think of contamination in my miniprep solution (ethanol, salts, and other possibilities).

I use the amersham sequencing kit for my preps and before resuspending my sequences (in high deionized formamide), the drying must be on room temperature !!!

hope that could help you.




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