I have been trying for months to clone a 4 kB fragment into a 6.4 kB BAC (I know it's very small for a BAC). I know my restriction enzymes used to prepare insert and vector are good (i.e drops out insert) and I know my ligation reagents are good (i.e ligates lambda-HindII markers). Lastly, I can transform bacteria with vector + insert ligation but always recover vector only when screening resulting colonies. Ultimately I am trying to construct a synthetic virus. Any advice/suggestions are welcome.
Is there any chance that one of your enzymes/buffers might have been contaminated with small amounts of nucleases? I once saw that happen in a lab where many researchers and pregrad students -some with really sloppy technique- shared their reagents. The restriction digests looked beautiful, but cloning never worked. After a very frustrating month the problem was eventually traced back to a nuclease-contaminated tube of Amersham's H 10X buffer.
Another thing. In my hands, the larger the fragments, the more important it is to avoid irradiating them with UV, even when using longer wavelength boxes. Are you gel-purifying the insert? If so, it might be better to skip the purification step altogether and simply ensure, during screening, that you are not recovering parental colonies. This is easy if the parental and recipient vectors have different selection markers, but even if they have the same marker, nothing stops you from recutting your ligation mixes with an r.e. that only cuts in the parental vector.
Hope it helps.
Simple question, do you dephosphorylate your vector? Are blunt-end or sticky end cloning?
I am sticky end cloning and have tried both dephosphorylating and not dephosphorylating the vector.
Dont use restriction enzymes, it sucks. Use gibson assembly and PCR amplify vector and insert.
Use Gibson assembly cloning. PCR amplify your insert with homology to the BAC backbone.
And try to switch to a conditionally amplifiable BAC. Then you can artificially increase the copy number and you avoid dealing with the low copy number. Just amplify from low copy number to high copy number and do a regular miniprep for the plasmid. Then you do not have to PCR amplify the backbone.
Recombineering can be a good option for you. For more information, take a look here: http://redrecombineering.ncifcrf.gov/
You possibly have incomplete digestion of the BAC and have some supercoiled in your digested preperation. Try running the digested BAC on an agarose gel and cutting out tand purifying the 6.4 Kb fragment before ligation.
By conventional cloning, try that the plasmid from which you are getting the insert have a direct antibiotic resistant than the BAC. If so, you can do all the cloning in solution (without purifiying the insert or the vector from the gel) and will give you much better results
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning