IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/24

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Raphaël
Minipreps : 4 minipreps of a 10ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL:

  • LacZ alpha (I732006)
  • RBS/GFP/term (E0240)
  • LuxR (C0062)
  • RBS/LuxI (K081008)
  • TetA(C) (J31007)


Restriction digest of the same minipreps as yestarday (concentration measured by nanodrop 07/23), around 3µg of DNA per reaction.

  • term/attC/mRFP : 42µl DNA + 5µl Buffer 3 10x + 0.5µl BSA 100x + 2µl XbaI + 2µl PstI
  • attC/term/attC/mRFP : 42µl DNA + 5µl Buffer 3 10x + 0.5µl BSA 100x + 2µl XbaI + 2µl PstI


Gel electrophoresis of the restriction digest products (future inserts)
Agarose gel 1.5%, 50V

  • Ladder 1Kb
  • attC/term/attC/mRFP : 25µl + 5µl lb
  • attC/term/attC/mRFP : 25µl + 5µl lb
  • -
  • term/attC/mRFP : 25µl + 5µl lb
  • term/attC/mRFP : 25µl + 5µl lb

Image:gel2407_1.jpg Image:gel2407_2.jpg
The absence of the band corresponding to the insert for attC/term/attC/mRFP raises some questions about the real nature of this plasmid...


Gel purification with the Quiagen kit.
All bands are eluted with 30μL of water.


Overnight cultures of the transformations from 07/23 (colonies on all plates):
2x20mL for each transformation

  • TOP10 pSB1A2 weak RBS -TetA(C) - Amp
  • TOP10 pSB1A2 med.RBS -TetA(C) - Amp
  • TOP10 pSB1A2 weak RBS -LuxR - Amp
  • TOP10 pSB1A2 med.RBS -LuxR - Amp
  • TOP10 pSB1A2 stand.RBS -LuxR - Amp


Nanodrop to determine the amount of DNA in the miniprep samples and after gel purification:

Sample name 260/280 260/230 ng/μL
t-attC-mRFP (gel purif.) 1.610.026.3
LuxI (miniprep) 1.92 2.30203.4
LuxR (miniprep) 1.902.30221.5
LacZ (miniprep) 1.91 2.33 299.0
TetA(C) (miniprep) 1.91 2.26 303.4
GFP (miniprep) 1.90 2.29 239.1


Same remark as yesterday, there is not enough DNA after gel purification to do a ligation... On the other hand, make minipreps with 10ml of pellet increases the amount of DNA in our initial samples.



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