Protocol Online logo
Top : Forum Archives: : Molecular Cloning

how to tell the transformation result? - (Feb/15/2008 )

after transformation i miniprep the cells and then digested them
after that i run a gel and the result is like this




(1) is the dna i digested
(2) is the orginal vector
ps: the (1)and (2)are on different lanes

it's hard to present the ladder so i use the original vector to compare here
plus, i have only one colony in my plate,
double digested
so i should see two bands on (1) but there is only one here,
i was confused what is that ? the dna in the colony survived on the media with ampiciline
many thanks


A photo of the gel would be useful. Is your original control plasmid also digested? If not, it may simply be that your new vector doesn't have an insert but is traveling through the gel at a different rate than the digested one due to supercoiling (of the uncut control). If you digested your control plasmid, what is the difference in size on the gel?


the vector is not digested, it's the original one, just put this to compare the band, no other use
i was confused with the only one band on lane(1)
why i only got one band on the (1)lane?
if the transformation was failed , then what is that band?
many thanks


It seems to me that your transformation was successful (thus the ampicillin resistance) but your ligation was not (I assume you are transforming a ligation, correct?). I think the difference you are seeing in the size is because one is digested and thus linear (lane 1) and the other is not digested and thus supercoiled (lane 2). I could be wrong though, hard to tell with no picture of the gel itself. How far apart is the lane 1 band traveling compared t the lane 2?


i would concure with Captain_DNA. Since the miniprep digest does not match what you expect, that single colony is not the right thing. In addition, a sucessful ligation should produce more then just one colony. You should expect 100s of colonies.