Vector digestion - (Feb/24/2008 )

hello
I m using pBS sk+ as vector to transform E.coli. but after purification and vector digestion, I got only my vector not my GUS construct.
can someone tell me where is the problem?
thank you

Maybe the cloning didn't work properly. Did you check your colonies by PCR?

There are many things which could have gone wrong in cloning. From restriction digest of vector and insert to amount of DNA, the ligation buffer and enzyme etc.

Look at each step critically to find the possible cause,

There are a thousand and one things that could have gone wrong. Form your selection of restriction enzyme, to your digestion time being too short or too long, to you T4 DNA ligase or buffer having gone bad, overdephosphorylation, insert being toxic, poorly PCR insert, poor transformation protocols, accidentally used the wrong antibiotic... etc.... so many things. (you get the idea)

Thus, tell us what you did in every possible detail. What is the nature of your ligation? What enzyme were used? How long did you digest? What test you have done. What controls? How many colonies do you have on your plate? If you have 10... then your transformation didn't work. How many colonies did you look at? What kind of your transformation protocol did you use?

thank you for your reply, so I m trying to make knock down construct.
I choose DNA sequence amd I cut it by 2 different enzeymes: SpeI/HindIII, XhoI/EcorI.
Then I cut GUS linker fragment by HindIII/EcorI.
I prepared also my vector pBS sk+, I cut it by SpeI+XhoI.
for ligation reaction I mix them with Ligation High (v2.0) enzyme, overnight at 12c.
then I mix the 5ul of igation reaction with 50ul competent cells and spread into LB plate+Amp, and incubation overnight.
after I got colonies, I make culturee and incubation overnight.
after purification i cut my vector with SpeI/XhoI, but I found only vector but not my DNA sequence+GUS.
so I don"t know where is the problem?,