I am a little stuck with a digestion/ligation that i have attempted a few times & hoping somebody can give me a few suggestions?
The vector and gene are being restricted with kpnI.
I get the correct bp band for the gene and cut out & gel purify.
The vector is treated with AP 1 hour & analysed by agarose gel. The vector yields a band of correct bp(~7500), but also a higher bp band (~10000), which is not present in the undigested sample. I cut out and gel purify the band corresponding to the linearised vector (~7500bp)
Ligations have been attempted at 1:10, 1:6, 1:3 and 1:2. Always alongside a negative control (just linearised vector/no insert).
I get colonies for 1:10 - 1:3 ligations and the negative control has none. Looks promising, but when i prep up these colonies and digest with kpnI again, i get religated vector and on agarose gel I observe the same as before - a band corresponding to the linearised vector (~7500 bp) and the higher bp band?!
Where am I going wrong?! Thanks for reading.