Cloning - Problems about cloning (Jun/12/2006 )
I am using pBAD (4092bp) as plasmide, Normally I can see 3 bands on the gel, but after its digestion with ecoR1 and dephosphorylation i only see 1 band, This vecteur I use.
My insert have about 2100 bp. After the ligation i did a migration on the gel and See 3 band:
1: about 1250 bp,
2: about 2100
3 over than 10000bp
Which would be the problem?
Thank very much
if i did not misunderstand, you see 3 bands when you load your vector on gel. this is normal, linear pen circle and supercoiled forms of plasmid migrates with different speeds. so when you digest your vector with a restriction enzyme, if digestion has worked, you will hope to see only one band (if your enzyme cuts the vector only once, of course) and that band corresponds to linear form of vector. its size should be 4092. check that.
coming to your ligation problem, what controls did you add to your ligation experiment? are you sure you could succesfully dephosphorylate your vector. the band over than 10kb may represent a concatamer which can occur in cloning with only one restriction enzyme?
if you tell more on your exp. and controls, i may help you better,
Thank you, in my control i did not add ligase and i can see 2 bands, vector and insert, my control, and 3 bands, in the ligation,
I don't understand why I see a band, which has about 1250 pb, and why my ligation didn't occur.
Is possible to avoid have a concatamer?