IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/07/23

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July 23, 2009

Genomic DNA prep, culture PCR

- PCR to check if GFP-Kan unsorted in correct section in the genome.

- Forward primers homologous to gene. Reverse is homologous to portion inside longtine cassette.

Note: will need to make more TE for satoe.

          50 mM tris pH 7.5
          10 mM EDTA

Reverse primer, use Kan LR

          - 520 bp + GFP fragment
          - 20 mM stock from Folu
          - dilute to 5 uM. start with 20 mM

25 ul + 75 ul dH2O

- After shaking of EtOH (step 23) will leave O/N to evaporate EtOH - PCR w/single colony. If doesn't work, will use genomic DNA

Gene/culture Code

           035-1             1
           35-2              2  
           35-5              3
           139-1             4
           139-2             5 
           49-1              6
           49-2              7
           186-1             8
           186-2             9
           213-1             10
           213-2             11
           236-1             12 
           236-2             13
           346-1             14
           346-2             15

PCR - 1 bead

- 2.5 ul primer 1

- 2.5 ul primer 2

- 19 uL dH2O

- culture spot

Run on cycle iGEM 6

Spot control 16

No Primers 17 (Use 35-5)

End of Day




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