Clone 2kb and 5 kb inserts into 6kb vector - Which order and vector:insert ration? (Feb/13/2007 )
I am trying to create a conditional gene targeting vector. My vector is around 6kb and the inserts that I want to clone in are 2kb and 5kb. I have to clone them both in. My question is which insert (small or big one) would you first clone and then what kind of a vector:insert ration would you use when you would be ligating.
Just do them next to each other? Use ligation ratio's of 1:1 to 1:5 or so, if you're digests are clean (no non-cut vector etc), you should get them easiliy. Are they blunt or sticky ends?
I clone these inserts into the same targeting vector but the cloning sites for these two inserts, in the targeting vector are seperated from eachother by neo marker and loxP sites.
Secondly, I am doing sticky end cloning.
Thank you for the suggestions.
I would clone in the smaller fragment first (2kb). I would use a 1:1 mol ratio betwen vector and insert. Keeping the concentration of each component as high as possible.
Later would clone in the 5kb fragment. I would use a 1:3 ratio between vector:insert. Again I would keep the concentration as high as possible.
Large vectors are a bit difficult to ligate. Furthermore, large vectors also do not bind to silicon column well (which excludes the use of gel purification columns), thus requiring electrolution to extract the DNA from a gel slab.
The upper limit for efficient DNA retention by nearly all columns is about 10kb. So, (6kb+ 2kb = 8kb) keeps the plasmid/vector size under 10kb.
6kb +5kb = (electrolution or cutting more DNA to make up loses on the column) AND (slight increased difficulty when trying to ligated the 2kb fragment)
General idea is as the vector size increases, the ratio of insert increases to compensate for increased dfficulty of ligation.