hi

plasmid after DNA miniprep It can be found in two forms usually , 80% super coil and 20% linear . DNA size marker is linear form and not suitable for compare the super coil size of DNA . also super coil DNA run faster in agarose gel than linear form of DNA .

good luck

Skipping the plating is not a good idea, first you might gt a mixture of recombined and not recombined plamids, for instance if you have an incomplete digestion (only one enzyme cut) you will have self-ligation and the empty vector as the smallest molecule will be preferentially uptaken by the cells. Second, you will have your recombined clone with cloned fragment;third you might have two vectors ligated together ....(large molecule -low transformation efficiency). so if you dont have any direct selection (drug resistance on your fragment) you should not skip the plating (even with direct selection I would not recommend it).

two bands might be an empty vector and your recombined vector (single colony would help), but also all plasmids have 3 forms (ccc, oc, lin) on a gel so after extraction you will always get more than a single band on a gel (usually 3 but sometimes much more if concatamers are formed).....

and you cant compare the size of linear ladder and not digested plasmid..... these are different forms they migrate differntly.....

Hi,

It seems your insert is not cloned in your vector. This is vector self ligation. Which Enzyme you are using for digestion ? Digest original vector ( base plasmid) and cloned plasmid using other single cutter (other than SalI and NotI) present in plasmid.