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From 2013.igem.org

colonies	Normal concentration	High concentration
control	11	60
Fnr in plasmid PSB1C3	0	2

We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control)

Primer and PCR

VF2, VR, Pfnr_up, Pfnr_down are 4 primes that we used for plasmid restriction. The primers, if the promoter fnr entre the plasmid successfully will amplify 3 sub-pieces with specific size. They are VF/VR, VF/Pfnr_down, Pfnr_up/VR.

So we had prepared 4(colonies)*3(amplification) = 12 PCR tubes.

• Dream Taq(5µg/µl):2µl
  
• Buffer (Dream Taq) 10X:10µl
  
• dNTP:2µl
  
• Primer (F/R;F/fnr_R;fnr_F/R):2µl+2µl
  
• H2O:82µl
  
• Total:100µl (volume for 4 tubes, so 25µl for each)
  

PCR programe

The PCR products was put on a gel of agarose 1.5% with BET (1.5%), migrated during 30 min at 100V. the picture analysis was be delayed to the next day.

Culture confirmation

We performed another colonies seeding. 2 colonies selected from each Petri dish were seeded in liquid medium with their corresponding antibiotic at 37°C under stirring during 1 night.