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pBluescript SK (-) - (Jan/31/2006 )

Hey guys!
I've attached a plasmid miniprep of pBluescript SK (-) which is size of 3kb...do you think this miniprep looks normal eventhough the plasmid runs a bit lower (around 2kb, the big band)!? Does it mean it is denatured? I've read in Quagen miniprep troubleshooting that sometimes the plasmid during plasmid prep can become irreversibly denatured and runs faster on agarose gel. What are these other bands?

-smoochiepie79-

well to be more sure of your prep you cand igest a small aliquot 1-2┬Ál in order to linearize and avoid ll boring supercoiled and relaxed forms of plasmid...

anyway in a standard prep you often see 3bands (normal relaxed and supercoiled) so assuming the 4th band is the one you've mentionned...

-fred_33-

Best thing to do indeed is a restriction digestion.

Also, the big band you're seeing should be the supercoiled plasmid, which migrates faster than linear, so it should be "below" 3 kb (though it is hard to tell where it should exactly be).

-vairus-

QUOTE (vairus @ Jan 31 2006, 05:23 PM)
Best thing to do indeed is a restriction digestion.

Also, the big band you're seeing should be the supercoiled plasmid, which migrates faster than linear, so it should be "below" 3 kb (though it is hard to tell where it should exactly be).


Thanks you guys! How do you think a denatured plasmid would look like, would it be like a big band or some kind of shear?

-smoochiepie79-

blink.gif i don't know blink.gif but very interested by a photo of the restriction analysis.

-fred_33-

QUOTE (fred_33 @ Feb 1 2006, 04:13 PM)
blink.gif i don't know blink.gif but very interested by a photo of the restriction analysis.


This is not a restriction analysis, it's a plasmid prep.smile.gif!

-smoochiepie79-

still interested by a picture... wink.gif

-fred_33-

QUOTE (fred_33 @ Feb 8 2006, 03:28 PM)
still interested by a picture... wink.gif


wink.gif

-smoochiepie79-