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plasmid extraction and purification - methods to extract and purify plasmid to get high purity super coil (Jan/25/2011 )

Hi all,
I am struggling 1 year to get super coil plasmid using giga prep protocol without kit. However, all the plasmid I got was not in the super coil form.
If any one has experience please share and advise. We thanks you a lot.

-amdo-

How do you know it was not supercoiled? Do you have nicks, or even double strand breaks?

-Rsm-

Rsm on Wed Jan 26 08:48:22 2011 said:


How do you know it was not supercoiled? Do you have nicks, or even double strand breaks?

I run gel electrophoresis using 1-10 kb ladder. My plasmid is pGL3 (around 5kb) and I found one or sometimes 2 bands behind the last band in the ladder (10 kb). My ladder is (GeneRuler™ 1 kb DNA Ladder, 250-10,000 bp):
http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm031-generuler-1kb
is my work correct?

-amdo-

amdo on Wed Jan 26 13:54:12 2011 said:


Rsm on Wed Jan 26 08:48:22 2011 said:


How do you know it was not supercoiled? Do you have nicks, or even double strand breaks?

I run gel electrophoresis using 1-10 kb ladder. My plasmid is pGL3 (around 5kb) and I found one or sometimes 2 bands behind the last band in the ladder (10 kb). My ladder is (GeneRuler™ 1 kb DNA Ladder, 250-10,000 bp):
http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm031-generuler-1kb
is my work correct?


Do you make a analytical restriction digest for your prep, or run the plasmid straight away? I find undigested plasmids rather difficult to analyze, all sorts of sizes.

-Rsm-

Actually I run staright forward without digestion. If I digest, can I differentiate is it super coil or nut, I have never try.
Thanks for help.

-amdo-