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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