Hi all,
I was doing plasmid double digestion and got a weird result. The uncut plasmid should be 7kb. I expected to see 3 bands--nicked, linear, and supercoiled. However, I only got one band that's higher than 7kb, which means they are all nicked??? I'm confused...
I later used this linearized plamid to do transformation and always got colonies on my negative control plate. I thought it might be the quality of plasmids that caused this issue, since the uncut plasmids showed weird bands.
Can anyone help analyze this issue?
It looks to me that your uncut plasmid is probably a dimer (or larger). This happens often and is not something to worry about. If you put your plasmid in a RecA+ strain you would observe a number of forms in the uncut plasmid population, including monomer, dimer, trimer etc as recombination occurs to both increase and decrease the multimer number. But since most cloning strains are RecA- the multimer length becomes fixed, normally a monomer but not always.
I do not know about plasmid dimers (never occured to me).
To me, it looks like you may be working with something else that what you are expecting. Are the SpeI and XbaI sites close to eachother ?
I recommend you to perform an analytic digestion (restriction mapping). Either pick an enzyme that cuts >3 times, or digest your plasmid with several compatible enzymes and verify if all of the bands are of the correct size.
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