Need help! - Problem with right insertion! (Feb/18/2011 )
I am trying to clone a 1,200 bp PCR products of PISTILLATA in the plants. I did directly clone the PCR products. Approximately half of the clones was white. When I check the size of the insert by PCR amplification, there was no right insert (50-100 bs of inserts). I ran the PCR products on the gel, and cleaned the right fragment from the gel. This time, the cloning did not work at all. Only 2-4 blue clones, nothing more on the plate. I did this test several times, but no success. Anybody can suggest any solution? Thank you.
What kind of cloning are you doing?
Is this a blunt end cloning?
Did you gel purify your DNA fragment? Did you use a column to purify the DNA?
What kind of vector are you using? Is this a commercial vector? How was the vector prepared? Did you gel purify the vector too? Did you dephosphorylate the vector?
How long did you dephos the vector? How long did you ligate for? At what temperature?
How large are you vector and insert? What kind of methods are you using to transform the plasmid into the cell?
The ligase and ligase buffer easily expire. Did you notice a strong smell with the ligase buffer? How old is the T4 ligase? Is anybody in the lab experiencing problems with the ligase buffer? Did you check on a gel to see if the insert and vector had ligated?
There are many reasons why a ligation could fail. So it is important to provide us with every detail of what you did.