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

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(Check PCR products)
(RBS screening (single digest))
Line 48: Line 48:
- 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
- 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
-
- Gel
+
- 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
==Check PCR products==
==Check PCR products==

Revision as of 05:36, 16 September 2008



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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

product concentration (ng/μL) 260/280 quantity of DNA to add to have about 300ng (μL) added SDW for single digest (μL)
RBS W1 57.1 1.57 6 11
RBS W3 54.1 1.53 6 11
RBS W5 15.1 1.85 17 0
RBS W 7 21.8 1.66 15 2
RBS W9 21.8 1.78 15 2
RBS W11 13.8 1.73 17 0
RBS S2 19.5 1.7 15 2
RBS S4 82.13 2.57 4 13
RBS S6 44.7 1.64 7 10
RBS S8 104.0 1.48 3 14
RBS S10 13.1 1.82 17 0
RBS S11 20.1 1.73 15 2

- 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

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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