cloning probelm - (Apr/02/2007 )

Hi all

I have a cloning problem; if someone could help.
I am using the topo TA vector to clone a 950 bp insert into it. Upon ligation and extraction of the plasmid i wanted to confrim by Ecor1 restriciton. However after gel electrophoresis i could not see my insert band on the 2% agarose gel.
I used aproxx 200ng of DNA with 1ul of Ecor1 enxyme, 1x buffer and remaining water.... where did i go wrong ?? should i try running them on 1% agarose.....

pls help

prats

---

2% TAE agarose gel is fine. No real need to change. (1.5% would be better then 1%)

As for the digest, 1ul of EcoRI, assuming this is 1ul of undiluted enzyme from the vial, that is alot of enzyme. How large is the digest 10ul...20ul?

I would suggest a digest of 0.5ul in 20ul volume (there is no real need to percipitate the DNA (gel looks better with percipitation but unrequired). Run the entire digest directly on the gel, use a lower voltage about 80mV.

As for DNA, you could cut abit more, try about 500ng upto 1ug. 200ng, the 900bp band may be too faint to see if your gel isn't clean (electroporation box is dirty, buffer is dirty)

If this is still at the screening stage, colony PCR might be helpful. If you have primers that can prime across the insert, or primer across the juntion between insert and vector, colony PCR might be an option.

---