Mathies:E COLI PCR

E. COLI PCR

Reagents

Templates: E. Coli K12 or O157

Primers: Primers specific to the KI#128 island on the K12 genome (forward primer: 5' - TTC GAT TAC ACG GAG TGC TGG GAA - 3' and reverse primer: 5' - CGT TGA TTT GCC GTT CCA TGT CGT - 3'; 259 bp product) and the OI#43 island on the O157 genome (forward primer: 5' - TGG CAG GAA GAG AGT GAC AGG - 3' and reverse primer: 5' - GGC CTT ACC CGT GAA CAG TA - 3'; 191 bp product) were designed to prevent any cross-amplification between strains. Forward primers were labeled with a 6-FAM dye on the 5-prime end (Integrated DNA Technologies). (Lewis 313 F1 Bottom Left “Lina”) *bleep*tail: Qiagen HotStarTaq Master Mix (400 µM each dNTP, 3 mM MgCL2, 0.1 units/µL Taq)

PCR-proof water

Protocol

1µl of each template

1µl 10µM of forward and reverse primers

12.5µl of Qiagen HotStarTaq Master Mix

10.5µl of water

Thermal cycling program: SAPCR

10 minute 95 °C (hot start step to activate the Taq polymerase)

29 cycles of 94 °C denaturation (30 sec), 60 °C annealing (30 sec), and 72 °C extension (30 sec)

5 minute 72 °C

Incubate at 4 °C before other analysis

Contact

• Eric Chu 22:25, 22 July 2009 (PDT):

or instead, discuss this protocol.