Conditions and cloning rates for kanamycin insertion into pBAD by IFPCa.

a)For comparability all data shown was generated in one parallel setup.b)The standard procedure and PCR conditions are described in the material and methods part. If not other mentioned, gel-eluted insert and plasmid derived from a mini-prep were used as templates. Normally only a very light or even no band is visible on an agarose gel when 10 µl of the inverse fusion PCR was loaded.c)Instead of plasmid an E. coli colony containing pBAD was used. pBAD is a high copy plasmid. Low copy plasmids will need lower dilutions for optimal IFPC performance.d)Since the (NH4)SO4 present in the PCR buffer inhibits phosphorylation by T4-pnk, one experiment was performed with 2 µl fusion PCR while the other one was prepared with 0.2 µl. 1,870 colonies were counted per 0.2 µl of fusion PCR, for comparison 9,350 colonies are the calculated colonies per µl of fusion PCR.