Restriction digests of the clone give the following sizes (kb): BamHI--6.1; XbaI--6.1; EcoRV--4.35, 1.75. Vector contains the following restriction sites (approximate kb from nt 1): BamHI--0.14; BglI--1.40, 1.63, 4.47, 4.59, 6.04; ClaI--0.49; EcoRV--0.65, 2.40; NcoI--0.11; PvuI--1.86, 4.72, 6.07; PvuII--1.89, 2.16, 2.25; XbaI--0.07. Mutagenesis is achieved through alkali denaturation of the plasmid containing a cloned insert, followed by amplification of the plasmid using an ampicillin repair primer and a mutagenic primer. Single stranded DNA can also be generated by infection with a helper phage such as R408 or M13K07 (ATCC 37468). Following in vitro synthesis of the second DNA strand, the plasmid should be grown in E. coli BMH 71-18 mutS or ES1301 mutS (to avoid repair of the mutation) and can be selected for ampicillin resistance. Clones can then be transformed into a suitable strain for propagation (E. coli JM109 - ATCC 53323) or expression (E. coli BL21 (DE3)). Several rounds of mutation can be achieved by including a tetracycline inactivation primer in the first round, so that tetracycline resistance can be restored and used as a marker for second round mutants. Best mutagenic efficiency was achieved using a 10 fold molar excess of mutagenic primer to ampicillin repair primer. Expression vector allowing site directed mutagenesis of cloned inserts and subsequent selection of mutants for ampicillin resistance. The following oligonucleotides can be used to repair or inactivate the vector markers: ampicillin repair, 5'-GTTGCCATTGCTGCAGGCATCGTGGTG-3'; ampicillin knockout, 5'-GTTGCCATTGCGGCATCGTGGTGTCAC-3'; tetracyclin repair, 5'-GCCGGGCCTCTTGCGGGATATCGTCCA-3'; tetracycline knockout, 5'-GCCGGGCCTCTTGCGGGCGTCCATTCC-3'. Constructed from pSELECT by replacing the lacZ region with a T7 promoter, under control of the lac repressor, and replacing the rop copy number control sequence with the lacIq repressor gene. |
