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Will self-ligated plasmid run faster than the unligated one? - (Jan/31/2007 )

I have this question, and got different answers from people in my lab. If I make a double enzyme cut on a plasmid and gel-purify the big band, then I self-ligate it. After running it on an agarose gel, should the ligated form run faster than the unligated form ?
Thank you!

-cathywill-

If you run a linearised plasmid and its supercoiled form, the supercoil version runs faster.

IF you digest a plasmid and then religate it and in comparison to the digested plasmid (still linearised), the religated one should run faster on a gel.

-scolix-

linear form runs faster on the gel.... unsure.gif am I confusing something? supercoil should be much higher than the linear on the gel.

-Kathy-

Hi Kathy,
Relative mobility of plasmid on agarose gel
I am with Scolix. Supercoil runs faster on gel as compared to linear plasmid.
Hope this will help.

-Niraj-

DNA is like those old phone cords

supercoiled is like when they get all wound up on themselves and become a big snarly knot, they coil up tightly and can go through the gel matrix

relaxed is spread out a bit more

linear is spread out even farther - picture a long phone cord stretched out, trying to wind it's way through something porous like a sponge. the supercoiled form, being all tightly wrapped up, will go through the 'holes' and 'gaps' in the matrix more easily and therefore faster

-aimikins-

thank you for the link. well that is strange. my cut plasmid runs always faster than the supercoil, even the lowest band of it. unsure.gif

-Kathy-

Yup, supercoil will travel faster. But sometimes my plasmid DNA has 4 different bands. Sometimes only 3, sometimes 2.

Actually how can I explain the inconsistancy? Behaviour of the individual plasmid?

forgot.... thanks for the link too. wink.gif

-timjim-

QUOTE (timjim @ Feb 5 2007, 06:07 AM)
Yup, supercoil will travel faster. But sometimes my plasmid DNA has 4 different bands. Sometimes only 3, sometimes 2.

Actually how can I explain the inconsistancy? Behaviour of the individual plasmid?

forgot.... thanks for the link too. wink.gif



HI timjim,

When we get four bands its most likely that the fourth extra band is due to improper alkali lysis step. Well the other three bands correspond to the three forms of DNA (supercoiled first followed by opencoiled followed by linear). Two bands u get when ur prep is relatively good without the linear form.

Cheers

-tuttu-

Hi tuttu (cute name by the way)

Thanks for clarifying. All the while I ignored the existence of different bands cause I thought it doesnt really make any difference once I cut it with RE.
Thank you very much.

-timjim-

QUOTE (Niraj @ Feb 2 2007, 08:16 AM)



Thanks for the link. But I'm a bit confused... wacko.gif
May I know the differences between "nicked", "covalently bonded" and "open circular"?

Thanks.

-why-