IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/13

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(RBS W screening)
Current revision (06:06, 16 September 2008) (view source)
(RBS W screening)
 
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- W11 with XbaI : uncut, self ligation
- W11 with XbaI : uncut, self ligation
 +
==Double check of RBS W6 and stock==
 +
- Grow RBS W6 in 10mL of LB with antibiotic
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Grow RBS S6

- Put the 10mL of LB with RBS S6 from yesterday into 200mL of LB with antibiotic

- Incubate at 37°C with shaking

RBS W screening

- Plasmid miniprep of RBS W2, 4, 6, 8, 10 9witouh endo wash buffer)

- Nanodrop

product concentration (ng/μL) 260/280 quantity of DNA to add to have about 300ng (μL) added SDW for single digest (μL)
RBS W2 42.4 1.62 8 9
RBS W4 35.1 1.61 10 7
RBS W6 41.0 1.67 8 9
RBS W8 72.8 1.61 5 12
RBS W10 51.0 1.60 6 11

- Single digest with EcoRI and XbaI

- Gel1

  • Lane2 : HyperladderI
  • Lane3 : RBS W2 cut with EcoRI
  • Lane4 : RBS W2 cut with XbaI
  • Lane5 : RBS W4 cut with EcoRI
  • Lane6 : RBS W4 cut with XbaI
  • Lane7 : RBS W5 cut with EcoRI
  • Lane8 : RBS W5 cut with XbaI
  • Lane9 : RBS W6 cut with EcoRI
  • Lane10 : RBS W6 cut with XbaI
  • Lane11 : RBS W8 uncut
  • Lane12 : supercoiled ladder


- Gel2

  • Lane2 : HyperladderI
  • Lane3 : RBS W8 cut with EcoRI
  • Lane4 : RBS W8 cut with XbaI
  • Lane5 : RBS W10 cut with EcoRI
  • Lane6 : RBS W10 cut with XbaI
  • Lane7 : RBS W11 cut with EcoRI
  • Lane8 : RBS W11 cut with XbaI
  • Lane9 : RBS W8 uncut
  • Lane10 : supercoiled ladder
  • Result

- W2 with EcoRI : cut

- W2 with XbaI : uncut, self ligation

- W4 with EcoRI : cut

- W4 with XbaI : uncut, self ligation

- W5 with EcoRI : cut

- W5 with XbaI : uncut, self ligation

- W6 with EcoRI : cut

- W6 with XbaI : cut (2 bands), transformation ok for RBS W6

- W8 with EcoRI : impossible to see

- W8 with XbaI : impossible to see

- W10 with EcoRI : cut

- W10 with XbaI : maybe cut, it seems to be 2 bands, but quite difficult to see

- W11 with EcoRI : cut

- W11 with XbaI : uncut, self ligation

Double check of RBS W6 and stock

- Grow RBS W6 in 10mL of LB with antibiotic


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