Bac recomb: cre recomb - (Nov/07/2007 )
hey,
I targeted a linear NEO cassette into Bac (CAM) plasmid in EL350 strain by recombineering (induction at 42).Then to remove cassette: I induced with arabinose at 32 and plated as usual on CAM or CAM/NEO and grew at 32. The numbers looked right.
Unind with Ara : similar no of colonies in CAM 12 and CAM 12/ NEO24
Ind with Ara : colonies in CAM12 and no colonies in CAM12/NEO24
Pcr results for the colonies.:
pcr for the cassette other than neo : looks fine
pcr for NEO : positive for clone BEFORE Ara Ind. But pcr for Ara induced colonies is positive but not as strong signal as in before Ara induction.
what is the explanation
Do i have more than one bac plasmid per cell and cre is partial ?? Do I pick one colony and do a repeated ara induction??
Other thing : the Ara ind colonies are growing in Neo24??
Yes, you do have more then one BAC per cell. And the recombinering will not target every BAC in the cell. So each BAC will have a mix population of native and enginneered BACs.
Did you conduct your entire experiment in a single step?
You should colony purify after the targetting by the NEO cassette. Pick a correctly colony, serial culture it in selection medium for a few days. Then replate on selection plate. Pick a few colony and make sure by PCR that they are pure, there is no indication by PCR that the native sequence remains. Once done, conduct the cre mediated removal of NEO gene.
And you should also colony purify after the cre mediated removal of said cassette. Is your Neo gene, a rpsl-neo fusion gene? Can you counter select?
And you should also colony purify after the cre mediated removal of said cassette. Is your Neo gene, a rpsl-neo fusion gene? Can you counter select?
thx- it explains some of my results whiich i thought were weird !!
If my neo is plain neo and my cre is partial , how do i get rid of neo plus bacs from mixed colonies ??
Hmm... are you replacing the chloroamphenicol gene (CAM) with the NEO gene? Does the CAM gene play any role in the recombination here? I am not quite clear. For now I will assume that the CAM (chloroamphenicol) marker is a spectator and does nothing.
As I had previously mentioned, if you do not have counter selection, you have to break your reaction into two halves.
In the first halve, the NEO cassette is transfected into cells and recombines with the BAC. Select for colonies which are neomycine resistent. You should have in mind that despite that being neomycine resistent, most of these colonies are composed of cells carrying both the Original BAC (OR-BAC) and the NEO cassette recombined BAC (NEO-BAC).
Pick 2 different isolates of these Neomycine resistent colonies and start culturing them in 2ml of LB+NEO/Kanamycine. Grow until culture becomes cloudy. (which may take 12 to 24 hours). The take a small innocculant from this culture and use it to innoculate a another fresh culture of 2ml LB+NEO. Do this for 3 to 7 days. If your plasmid is very large or unstable culture in a richer medium like SOC and lower the growth temperature to 30 Celsius).
Then spread said culture onto LB+NEO/Kan plates, such that you can isolate single colonies.
This whole lenght process is to purify your cell from OR-BAC. Since BACs are segrated randomly between daughter cells when the mother cell devides, do this enough times, and with luck you can isolate a cell which is pure.
The second halve is the Cre exposure.
Again the same happens. This time you will have plate the cells and pick single colonies and test for Neomycine resistence. If they are sensitive to neomycine/(or you can use kanamycine), it means that the NEO gene has been excised. And that is the colony that you want.
AS you can see without a counterselectable marker like (rpls-neo) the experiment become a headache.
Thanks a lot for the details.