problem with restriction digest - I have an insert, but no vector.... (Jan/24/2008 )
I am trying to clone an insert into a Pet-30 vector. I generated the insert by PCR adding on NdeI/XhoI ends. I cleaned up my pcr using the Qiaquick PCR purification. I digested my product (using Nde/XhoI) for about 3 hrs @37 deg. When I run the digest on a gel, I see my insert (which appears to be the right size), but not the vector. Help?
What vector?
After you purified your pcr product you'll have hardly any of the plasmid you used to amplify that fragment from...
Maybe I got things wrong....
You would have too little of the DNA template to actually see it when you run a gel.
werll did you mean you can't see the pet 30 vector after preparation ?
what's the starting amount of vector DNA ? did you load 1µl of undigested vector too ?
i have the opposite results with u
i can see my vector but cannot find the insert...
i can see my vector but cannot find the insert...
how much of the miniprep DNA did you digest? You need to have a particular amount of DNA in order to see fragments which are being digested out.
eg. if your vector + insert is 5kb and your insert is 500bps. If you digest 100ng of this, you will get ~90 ng of vector and 10ng of insert. And 10ng might be easily missed because of the low concentration.