Construction of Cysı Ala RsrA mutants, by long PCR mutagenesis. Dan White 11/01

Cys codons replaced with gcc ala codon.

Tube numbers in ( ) for stuff in Chalkie box.

Primers :

RSRA3ALA : gat cga gga ttt cgc tgc aat ccg tct cgt gcg gct ctc cgg cgc tca tat gg (11)

RSRA31ALA : atc tct acg agt ttc tcg aca aag aga tgc cgg act cgg acg ccg tga agt tc (12)

RSRA11F : ctc gat cat ctc tac gag ttt ctc (13)

RSRA11R : gat ttc gct ggc atc cgt ctc g (14)

RSRA41A : cga ggc ctc ctc gaa gtg gtg ctc (15)

RSRA44A : ccc gcc ctg gag aag tac ggg ctg (16)

NB RSRA311ALA primer was incorrect and was discarded.


Constructs:

pUC constructs. These were made by long pcr with pfu polymerase with the mutagenic primers  shown. PCR  pruduct gel purified, then self ligated in a mini sonicator at 4oC with high conc ligase for 20 min. Products were transformed into DH5_ and plasmids screened by sequencing for desired mutations, with universal and reverse primers.

(1) rsrA in pUC 3C, 11A, 31C, 41A, 44A, 61C, 62C.
template pMT3000::rsrA4CıS+A, primers RSRA3ALA + RSRA31ALA

(2) rsrA in pUC 3S, 11C, 31S, 41A, 44A, 61C, 62C.
template pMT3000::rsrA4CıS+A, primers RSRA11F + RSRA11R

(3) rsrA in pUC 3S, 11A, 31S, 41C, 44C, 61C, 62C.
template pMT3000::rsrA4CıS+A, primers RSRA41A + RSRA44A

(4) rsrA in pUC 3S, 11C, 31S, 41C, 44C, 61C, 62C.
template (2) (pMT3000::rsrA 113), primers RSRA41A + RSRA44A

(5) rsrA in pUC 3C, 11C, 31C, 41A, 44A, 61C, 62C.
template (1) (pMT3000::rsrA a14), primers RSRA11F + RSRA11R

(6) rsrA in pUC 3C, 11C, 31C, 41C, 44C, 61C, 62C.
template (5) (pMT3000::rsrA zr2), primers RSRA41A + RSRA44A


pET15b constructs (Nde1 BamH1 subclones from pUC constructs)

(7) pET15b rsrA 3S, 11C, 31S, 41A, 44A, 61C, 62C. rsrA fragment from (2)

(8) pET15b rsrA 3S, 11A, 31S, 41C, 44C, 61C, 62C. rsrA fragment from (3)

(9abcd) pET15b rsrA 3C, 11C, 31C, 41A, 44A, 61C, 62C. rsrA fragment from (5)

(10abcd) pET15b rsrA 3C, 11C, 31C, 41C, 44C, 61C, 62C. rsrA fragment from (6)