transformation - random genomic inserts (Oct/31/2006 )
am interested in cloning lesser than 500 bp restriction enzyme digested genomic dna fragments into my vector 9 kb long cut with the same enzyme. i tried transformation thru calcium chloride mediated method and electroporation too but everything failed. are there any suggestions as to any other improvements and special care to be taken while handling such plasmids?
any suggestions will be of great help.
Do you need to dephosphorylate your vector (a single cut or 2 REs)? When you said that you wanted to clone <500bp RE gDNA fragments, was the fragments obtained thru gel isolation & purification of digested gDNA?
You can try increasing the DNA for RE rxn 'coz the final purified DNA yield will be significantly less. Normally 10 ug to 20 ug would be suffice. I will use as much of the purified DNA for ligation w/o obtaining its conc. nor determining the vector:insert ratio. 100ng to 150ng vector seemed suffice for each ligation with as much insert DNA that can be accommodated in the ligation rxn.
Did you include a positive ctrl for transformation?
i do a phosphatase treatment. it is asingle enzyme reaction.
i isolate the genomic dna fragments thru gel isolation, purify it and quantify it. i have tried using 100 ng of vector.
i always have apositive control for transformation and it always worked.
I believe your ligation is probably not working, due to the low amount of insert DNA.
When that happened to me, I just digest more (15 to 30 ug) gDNA, load into a large well (taped the well comb with adhesive tape to "bridge the gaps between wells"), then cut it out. Later use as much of the purified DNA for ligation without bothering to look at the vector:insert ratio [Normally too low to register anyway].
If gDNA is limited, maybe you can introduce adapters to the purified gDNA fragment and PCR amplify them? I've read it somewhere but can't recall where.