IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/12

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RBS screening (single digest)
- Plasmid miniprep 6 colonies of RBS S and 6 colonies of RBS W (without endo wash buffer)

- Nanodrop

- Single digest : with EcoRI and XbaI, add SDW and DNA (according to the previous table, 300mg of DNA), 2μL of fast digest buffer, and 1μL of enzyme

- Gel1


 * Lane2 : HyperladderI
 * Lane3 : RBS W1 cut with EcoRI
 * Lane4 : RBS W1 cut with XbaI
 * Lane5 : RBS W3 cut with EcoRI
 * Lane6 : RBS W3 cut with XbaI
 * Lane7 : RBS W7 cut with EcoRI
 * Lane8 : RBS W7 cut with XbaI
 * Lane9 : RBS W9 cut with EcoRI
 * Lane10 : RBS W9 cut with XbaI
 * Lane11 : RBS W9 uncut
 * Lane12 : supercoiled ladder

- Gel2


 * Lane2 : HyperladderI
 * Lane3 : RBS S4 cut with EcoRI
 * Lane4 : RBS S4 cut with XbaI
 * Lane5 : RBS S6 cut with EcoRI
 * Lane6 : RBS S6 cut with XbaI
 * Lane7 : RBS S8 cut with EcoRI
 * Lane8 : RBS S8 cut with XbaI
 * Lane9 : RBS S11 cut with EcoRI
 * Lane10 : RBS S11 cut with XbaI
 * Lane11 : RBS S4 uncut
 * Lane12 : supercoiled ladder


 * Result



The size of our plasmid is about 3000bp (ok on the gel)


 * RBS W

- W1 with EcoRI : cut

- W1 with XbaI : uncut, self ligation

- W3 with EcoRI : cut

- W3 with XbaI : uncut, self ligation

- W7 with EcoRI : impossible to see

- W7 with XbaI : uncut, self ligation

- W9 with EcoRI : cut

- W9 with XbaI : uncut, self ligation


 * RBS S

- S4 with EcoRI : cut

- S4 with XbaI : uncut, self ligation

- S6 with EcoRI : cut

- S6 with XbaI : 2 band, this plasmid is partially cut!!! Transformation with RBS S

- S8 with EcoRI : cut

- S8 with XbaI : uncut, self ligation

- S11 with EcoRI : cut

- S11 with XbaI : uncut, self ligation

Double check of RBS S6 and stock
- Grow RBS S6 in LB with Cm35

Check more RBS W
- Grow RBS W2, 4, 6, 8, 10 in 10mL of LB with antibiotic

Check PCR products
- Gel (2μL of each product)


 * Lane1 : λ ladder
 * Lane2 : agrA
 * Lane3 : agrB
 * Lane4 : agrC
 * Lane5 : rep
 * Lane6 : pSB4C5 1
 * Lane7 : pSB4C5 2
 * Lane8 : Pxyl
 * Lane9 : Ppac
 * Lane10 : RBS S
 * Lane11 : RBS W
 * Lane12 : HyperladderI


 * Results



- agr B : 2 bands?

- agrC : ok

- rep : small band but good size

- backbones : ok

- promoters : ok

- RBS : nothing


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