NICKED DNA - (Jan/08/2010 )
I did miniprep for GABAA receptor after (fresh) transfrmation subunit and I got these bands.
Earlier any year before when I used to do the minipreps, I used to get the bands near 6000 bp.
Now I did the transformation with that same sample but i am getting the bands on the top of 1kb ladder.
What do you think that during this one year the plasmid DNA circular form has changed in to Nicked one?
Or I am loosing the correct plasmid.. I have done it almost 20 times but I never got that 6000 bp plasmid recently.
Now I am digesting it with restriction enzyme to see if ITS CUT OR NOT.
wHAT DO YOU THINK OF THIS. pICTURE OF THE GEL ATTACHED.
looks like degraded plasmid!
There is a minor band, to my opinion supercoiled DNA, beneath the major band, linear DNA. Upon the major band there is another minor band that looks like open circular DNA (single strand nicked DNA). This is what plasmids should look like ...3 bands. In your case it seems like the plasmid was nicked by something (could be due to repeated freeze-thaw cycles or some other means).
My hypothesis can be evaluated easily ...how large is your plasmid ...if the major band is realy linear DNA than it should be exactly the size of plasmid. How large is your plasmid DNA?
Its about 7000bp.