User:Meng Xiao He/Notebook/fall08/2008/11/19

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One primer Phusion PCR of suicide vectors

 * extension time: 4:30
 * annealing temp: 52C
 * primer: 1ul (in 25ul of rx) of M13F primer from TOPO kit
 * cycles: 50
 * DNA (old minipreps of cbb, cco, Duo): 2ul each
 * for control: TOPO A

Confirmation of pure cultures

 * Phusion PCR new TOPO cco, pER-cbb, pER-cco, pER-Duo#5, 7 colonies of TOPO C plate
 * Rx: 5ul mastermix, 4.3ul water, 0.5ul primer pair, touch of DNA/cells
 * Conditions: 2min initial denaturation at 98, 50s extension, annealing at 57C, 30 cycles

Loss of plasmid

 * Streaked out cultures of WM3064 on LB-DAP-agar, inoculate fresh LB-DAP liquid culture


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