IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/15

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Check big stock of RBS S and W

  • We want to check our big stock of RBS S and W, with all digest enzymes

- Plasmid miniprep RBS S6 and RBS W6 (from 200mL flask)

- Nanodrop

product concentration (ng/μL) 260/280
RBS W2 90.3 1.70
RBS W4 124.0 1.80

- Single digest with EcoRI, XbaI, SpeI and PstI

- Gel

  • Lane1 : HyperladderI
  • Lane2 : RBS S6 cut with EcoRI
  • Lane3 : RBS S6 cut with SpeI
  • Lane4 : RBS S6 cut with PstI
  • Lane5 : RBS S6 cut with XbaI
  • Lane6 : RBS S6 uncut
  • Lane7 : RBS W6 cut with EcoRI
  • Lane8 : RBS W6 cut with SpeI
  • Lane9 : RBS W6 cut with PstI
  • Lane10 : RBS W6 cut with XbaI
  • Lane11 : RBS W6 uncut
  • Lane12 : supercoiled ladder
  • Result

- Even no band for the uncut plasmid. There is a problem with plasmid miniprep, we will try again with less (more diluted) cells for plasmid miniprep.

- New plasmid miniprep (the pellets from 20mL of LB is diluted into 1.2ml instead of 0.6)

- New single digest

- New gel (same lanes than before)

  • Result

- One band for everything, but the same size than the uncut plasmid. Moreover, this uncut plasmid should be 3000bp, and here, it is more. So, we have a problem in the growth of cells, we will try again and check the 10ml tube which is used to inoculate the big flask and also the big flask.

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