I have cloned my insert into a TOPO-vector.
Then I digested with 2 restriction-enzymes that targets sites flanking the insert in the TOPO-vector.
I was expecting to get 2 bands (one band being the vector, and the other smaller band the insert).
But what I got was 1 single big band. So probably the digest didn't work.
Could that be because I used 0.3ul of restriction-enzymes instead of 1ul for each 1ug DNA? My supervisor told me to use 0.3ul to save the expensive reagent.
Despite the results from the electrophoresis, we now want to send it for sequencing. And I have been told different things.
a) I haft to construct primers for each 700bp region of the construct (overlapping ones) and ones I get them, I will send them together with the TOPO-construct for sequencing.
b )I don't need to design anything. Just send the TOPO-construct to the company that does the sequencing
Which one is it now? People are saying different things here (I am gonna talk with my supervisor, but I want to get some input from here as well)
Edited by Biologystudent, 08 April 2014 - 06:04 AM.