Ligation reaction gel picture - (Mar/29/2007 )

As a followup to my previous post, I started over the cloning [I am trying to clone shRNA sequences (~50bp) into a lenti vector (linearized runs at 7-8 Kb, circular runs at ~4-5 Kb). Because I had troubles with my cloning before, this time I run what was left of the ligation reaction on a gel to see if I have a ligated product.

I attach the gel picture. Lanes 1-7 are my ligation reactions with the inserts, Lane 8 is the control ligation with no insert. Lane 9 is a circular lenti vector with a similar insert inside (not a ligation reaction though, the mini-preped vector). As you can see, I can see inserts down below and I can see what seems to be linearized vectors. I dont get any band similar to Lane 9, which indicates that the insert has not gone in (not sure whether insert gets incorporated but the vector remains linear). What I wanted to ask if you can always see the ligated plasmids by running a gel of the ligation reaction. I am also a bit confused about the other band on the top.

The ligation reaction was at 10ul total volume, using 1 ul of T4 ligase and 1ul of buffer. I used 1ul (0,5 ug) of linearized vector and 2 ul annealed oligos (60ng/ul each). I run the ligation overnight at RT.

Could the amount of plasmid with insert formed be too low to detect?

Any help would be greatly appreciated!

anyone??