cloning large inserts in large vectors - (Dec/21/2006 )
I have a rather general question, maybe you can help... I noted that when using a rel. large backbone vector (12kb, binary plant vector) and a rel large insert (3.6kb) the efficiency of my cloning decreases very rapidly!
I cut both vector and insert (with 2 different RE (BamHI & AscI) and very often I do not get a single colony! (which means, i guess, that both enzymes cur well, since no religation of empty vector occured...)
I usually do 1:3 V:I ratio / Ligation 16°C ON (basically standard...)
So: Are there any helps/tricks/secrets about efficient "large vector/insert clonings"?
What are your experiences when using things in a similar range of size? What procedures do you apply? V:I ratios...? Ligation temp & time...? special secrets...?
Thanks in advance!
Low concentrations favor circularization, and this is especially important for long fragments. Aim for 1-5 ng/ul in the ligation. Also, the V:I ratio should be 1:1 molar. You need to be more careful than normal about UV exposure of the fragments. Where is the insert coming from? If from PCR, make sure the enzyme cleanup is very very good prior to digestion. You can save time by ligating at RT for 10 minutes with normal ligase. Transformation could be either chemical or eletroporation at this size. You might want to use a control transformation with your uncut plasmid to verify that transformation is working with large plasmids. Also, don't use too much ligation in the transformation. 5% by volume is a good place.
Sometimes overdigestion could lead to the ends being chewed up. And therefore one would not have any background bcoz they cannot ligate anyways. Take care abt overdigesting.
Also it helps for larger plasmids to use more DNA for ligation. I have done ligation where I have used vector 100-200 ngs and 1:3 or 1:5 ratio of insert. You could try this as well.
Good Luck !!!
the only thing I have to add is to use supercompetent cells. To get really good number of colonies from the transformation, it is best to go via electroporation. I use genehog. It is rather expensive but it gets the job done.
Have cloned roughly 5 kb fragments into 10 kb vector backbones (containing viral LTR's,so even more tricky).
My advice would be to never overdigest. RE's come pretty concentrated as it is. I also dephosphorylated my vector (with alkaline antarctic phosphatase, which is less agressive than CIP).
Do not expose your DNA to UV, use UV-free kits or just run a lane on the gel to visualise on your transiluminator and do not expose the DNA you're going to cut out to UV.
And, use supercompetent cells, I used omnimax competent cells (chemically competent) from invitrogen. For 3 different clonings got the right colony the first time. Make sure the DNA is really clean after purication (check your A260/280 ratio!) and proceed using only little DNA (volume-wise) in your ligations.