short oligos ligation and transformation - (Aug/30/2007 )
I have a very serious problem with my cloning.
I have a vector containing two genes with app. 35 bp linker between them. The linker has two restriction sites in it's ends: SalI and SacI and a BamHI site in the middle of it. I need to change this linker to another one (that is a site of interest from some gene). I ordered two ss complementary oligos that on annealing create a competible SalI and SacI sticky ends and the BamHI site disappears.
I digested the vector with SalI, SacI and BamHI (in order to get reed of those that are only digested by one RE) and purified the vector.
Now I need to ligate my new insert with the vector and to transform it to competent E. coli cells.
I did it several times (I also run a control of the vector self ligation).
I did get many colonies on the plate, (a lot on the ligated and a few in the control), how ever the PCR check of the colonies was unsuccessful.
I did a mini prep of about 12 colonies from the insert containing plate and digested it with BamHI (If it digests - no insert, if it doesn't digest - there is an insert) I didn't get any digestion in any one of the colonies (however, it didn't digest the self ligated plasmid either).
Now I really don't know what to do.
Thanks a lot.
i think the method you use is not the best one...
Do you have the vector ang genes without the insert on them ?
i attempted to get rid of a linker once and did not succed. I just get back little more earlier and redo the insertion on the plasmid with no linker.
No. I don't. I received the plasmid with the old linker as it is.
I just don't understand where is the problem with what I am doing.
Some things to check: When you ordered the oligos, were they phosphorylated? If not, the ligation will be more difficult (still perhaps possible). You can anneal the oligos and treat with PNK to phosphorylate them.
An alternative (perhaps easier) is to PCR the entire vector around the circle with a pair of primers with your choice of cloning sites inserted into the 5' end of the primers. Don't forget to add a few bases of 5' overhang to allow efficient cutting of the RE sites.