I am to clone a gene into a vector.
1) I mini prepped the gene to be inserted -----> ran a PCR with primers flanking the gene -----> digested the ends of the PCR-product to create compatible ends
2) I ran a electrophoresis of the vector... and got 1 single intact band.
I then digested the vector. and electrophoresis showed 2 separate bands (bigger band was cut vector and smaller band was the excised unwanted fragment). I cut out the vector from the gel and purified it
3) I ligated the vector and insert. 1:3 ratio gave me 13.5ng insert to 45ng vector. Which based on their separate concentrations are equivalent to close to 1mikroliter insert + 2mikroliter vector.
4) I transformed bacteria with this ligation-sample. But I got no growth.
5) I ran a electrophoresis of my vector backbone and it showed NO BANDS at all. So assumably, I lost the vector during the purification. Right? Is that a correct interpretation?
6) I also ran a electrophoresis of the insert and it showed 2 bands when I was expecting 1 band. So assumably, the insert had been cleaved. Right? Is that a correct interpretation? But how? Where and how did this unwanted cleavage occur?
7) I measured the vector again after all this and the nano-drop now gave me a reading that was TWICE as high as before when I did the experiment. WHAT?????!!!
Can someone please ease my mind and tell me what they think of all this?
Edited by Biologystudent, 21 March 2014 - 04:31 AM.