Miniprep DNA - (Mar/03/2008 )
hello
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.
I usually get 10 to 12 colonies by plate.
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?,
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.
I usually get 10 to 12 colonies by plate.
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?,
HI!
i don't understand the experiment' Do you have checked the sequences you are working?. How do you know that they are efectly cut it?. how do you do the
digestion? All together?, What about the buffers ?. Do you purified the products before ligation reaction?
please give us some details, !!
good luck
Could you go step by step over the digestion of different DNA. Also did you verify the amount of DNA used for ligation?