I am trying to cut my vector with XhoI and SpeI. I setup a digestion reaction like this:
3.3 ul template (1ug)
2 ul 10x NEB Buffer 4
2 ul 10x BSA (I prepared this by diluting 100x stock in water, not 1X buffer)
11.7 ul H20
Incubation 37 C for 1h, inactivation 65 C for 20 min.
Then I run on a gel the cut and uncut vector.
In each lane, I got two top bands that were quite faint, definitely not representative of 1ug. I also got some fat bright bands but these were very small size, they run past last band of ladder.
So my questions are:
Why am I seeing such a small amount of vector and backbone? I re-speced the template and im definitely loading 1 ug.
I dont see an insert or even linearized vector released. But I get with such low amounts maybe it's not visible? I think the bands suggest some cutting takes place, cause cut and uncut top bands are slightly different size.
What are the fat bottom bands? Is it possible it's all star activity and the whole template gets chopped up in small fragments. I think it's unlikely with those amounts of enzyme. Also, this band is present in the uncut vector!
I am beginning to suspect there is something fundamentally wrong with my template. Any suggestions? :/
Edited by biobio, 19 September 2009 - 03:25 PM.