Uncut plasmid DNA - (Nov/26/2006 )

Wheh uncut plasmid DNA is separated using agarose gel electrophoresis swil there just be 1 band visble of supercoiled DNA or 3 bands visible of open cirlce, linear and supercoiled?

Thanks

-Sarah_30-

QUOTE (Sarah_30 @ Nov 26 2006, 11:31 AM)

Wheh uncut plasmid DNA is separated using agarose gel electrophoresis swil there just be 1 band visble of supercoiled DNA or 3 bands visible of open cirlce, linear and supercoiled?

Thanks

Including possible dimer of supercoiled DNA, it can be 2-4 bands.

-genehunter-1-

Generally 3 bands

Highest - nicked or relaxed circular plasmid
Middle - linear
Lowest - supercoiled

Then you can get all those other dimer combinations and they are higher than the nicked or relaxed cirular plasmid band.

-killerkoz17-

QUOTE (killerkoz17 @ Nov 27 2006, 02:49 AM)

Two of my miniprep DNA. Left is a GOOD one in which you have a major supercoiled DNA and on the right is a bad miniprep having all kinds of DNA mentioned in the above quote!

-Calvin*-

ok......may i ask a qn: why one may found the 3 forms although the plasmid is considered as "uncut"!!!

-strawberry-

QUOTE (strawberry @ Nov 27 2006, 12:19 PM)

ok......may i ask a qn: why one may found the 3 forms although the plasmid is considered as "uncut"!!!

Because there was just not enough love... so the DNA don't love you back.

Basically the operator took too long to complete the DNA extraction and subsequent deproteination and/or has treated the DNA too harshly, resulting in much breaking of the plasmid.

Too much vortexing and/or took too much time to remove all the nucleases.

-perneseblue-

aha, so it's just related to the operation itself.. thank u

-strawberry-