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plasmid extraction and purification


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4 replies to this topic

#1 amdo

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Posted 25 January 2011 - 08:55 PM

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.

#2 Rsm

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Posted 26 January 2011 - 12:48 AM

How do you know it was not supercoiled? Do you have nicks, or even double strand breaks?
I got soul, but I'm not a soldier

#3 amdo

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Posted 26 January 2011 - 05:54 AM

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...1-generuler-1kb
is my work correct?

#4 Rsm

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Posted 26 January 2011 - 07:32 AM


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...1-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.
I got soul, but I'm not a soldier

#5 amdo

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Posted 26 January 2011 - 07:39 AM

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.




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