How big is big in terms of the gene of interest insert in plasmid? - (Dec/07/2011 )
Was wondering if a 16kb insert into a 3kb commercial vector is considered as "big" in terms of molecular cloning?
If so, are there any additional steps needed to be done to transform the gene of interest into competent E.coli? Would the strain make a difference?
i would definitly consider it "big"!
...you can try using different ligases like the Takara Long Ligase ...for details see here!
Additionally, there are different E. coli strains that work significantly better wither larger plasmids ...some say DH10B (you can also go for STBL, SURE or GeneHog cells) is good for large plasmids ...in general it is believed that strains with the deoR mutation (like DH10B) tolerate larger plasmids better than those without. Another genotype that is associated with increased uptake of larger plasmids is hte.