4 replies to this topic

[amdo]

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.

[Rsm]

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

[amdo]

Rsm, on 26 January 2011 - 12:48 AM, 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...1-generuler-1kb
is my work correct?

[Rsm]

amdo, on 26 January 2011 - 05:54 AM, said:

Rsm, on 26 January 2011 - 12:48 AM, 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...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.

[amdo]

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.