Sprinting fragments? - (Dec/21/2007 )
Here is the story:
Been trying to insert a pcr fragment into my vector, the [insert] should look like this after cloning is done (BB=backbone):
The Xba1-Xba1 product is 188 bp and I digested the plasmid with Xba1 but I couldnt locate it the first time I ran the gel to completion.
So, this time I stopped the gel (0.7%) half way through to see whats going on.
Here is a link to an image:
Left most lane is the 1Kb ladder ( http://www.neb.com/nebecomm/productfiles/7...1_v1_000034.gif )
and the other lanes are 4 independent colonies.
Could the bottom fuzzy band be the 188 bp band? any other suggestions? Thanks and season's greetings all!
to visualise a 188bp fragment, you will have to run a 3% agarose gel, possibily cutting double the amount of plasmid DNA that you might normally use.
I would also suggest that you use a ladder with smaller rungs (100bp, 200bp, etc) to use. The smallest rung on your ladder is 500bp, it won't be possible to tell for certain if your tinny fragment is around the right size or not.
Could you not use some other combination of restriction enzyme? One that produces fragments that is easier to visualise. Also do not XbaI can be dam methylated. Make sure your XbaI sites are not effected by methylation.