IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/19

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Aleksandra
Restriction digest Using the minipreps from 07/16
17 µL water + 10 µL miniprep + 0.5 µL BSA 10x + 1 µL each enzyme; incubation at 37°C for 1 hour

  • attC - SpeI and PstI
  • term-attC-mRFP - XbaI and PstI
  • term (B0015) - EcoRI-HF and XbaI
  • RBS/GFP/term (E0240) - XbaI and PstI
  • RBS/LuxR (J37033) - XbaI and PstI
  • pLux (R0062) - SpeI and PstI
  • RBS/LuxI (K081008) - EcoRI and SpeI
  • Pstrong (J23119) - SpeI and PstI
  • Pmedium (J23110) - SpeI and PstI



Gel electrophoresis (agarose 0.8% w/v, EtB)
2 gels, in each well 17 µL digestion product + 3 µL loading buffer 6x

  • ladder 3 µL (new ladder, 3 µL corrsponds to 0.5 µg DNA)
  • attC
  • t-attC-term
  • pLux (R0062)
  • RBS/GFP/term (E0240)
  • Pmedium (J23110)
  • Pstrong (J23119)
  • RBS/LuxR (J37033)
  • RBS/LuxI (K081008)
  • terminator (B0015)

File:Gel1uncut.jpg
Cutting out the correct bands

File:Gel1cut.jpg


  • ladder (not a precise quantity : see the second ladder)
  • attC
  • t-attC-term
  • pLux (R0062)
  • RBS/GFP/term (E0240)
  • Pmedium (J23110)
  • Pstrong (J23119)
  • RBS/LuxR (J37033)
  • RBS/LuxI (K081008)
  • terminator (B0015)
  • ladder 3 µL (new ladder, 3 µL corrsponds to 0.5 µg DNA)


Cutting out the correct bands

File:Gel2cut.jpg
The J37033 biobrick (LuxR) never seems to work. Each time we cut it with the enzymes, we don't get a 700bp band as expected. Maybe the biobrick is wrong? Aleksandra ordered primers to amplify the sequence (the primers are on the plasmid backbone flanking the prefix and the suffix). We'll try to get this part from the 2007 distribution plates and also assemble the RBS/LuxR ourselves from 2 parts.


Raphaël
Gel electrophoresis (agarose 0.8% v/w, 100V)Checking the amount of DNA in the miniprepf from 07/17
5 µL of miniprep

  • ladder 3 µL
  • attC
  • term-attC-mRPP
  • pLux (R0062)
  • RBS/GFP/term (E0240)
  • Pmedium (J23110)
  • Pstrong (J23119)
  • RBS/LuxR (J37033)
  • RBS/LuxI (K081008)
  • term. (B0015)
  • KanR (P1003)

File:Miniprepsof5ml.jpg


Théotime
Gel extraction of the bands cut from the gel just before, using the new Qiagen gel extraction kit.


Ligation Using the fragments purified from the gel electrophoresis.
15 µL insert(I) + 5 µL vector(V) + 6 µL water + 3 µL T4 ligase buffer 10x + 1 µL T4ligase. Ligation at 37°C for 45 mins

  • attC (V) + t-attC-mRFP (I)
  • pLux (V) + RBS/GFP/term (I)
  • Pm (V) + LuxR (I)
  • Ps (V) + LuxR (I)
  • term (V) + LuxI (I)


Transformation Chemical transformation of the TOP10 bacteria with 15 µL of the ligation products. Plating with the corresponding antibiotic:

  • attC-t/attC/mRFP - Kan
  • pLux-RBS/GFP/term - Amp
  • Pm-LuxR - Amp
  • Ps-LuxR -Amp
  • term-LuxI - Amp




Raphaël
Transformation Chemical transformation of the TOP10 bacteria with the biobrick plasmids from the 2010 distribution plates. Plating with the corresponding antibiotic:

  • I732006 (LacZ) - Amp
  • B0031 (weak RBS) - Amp
  • B0032 (medium RBS) - Amp
  • B0034 (standard RBS) - Amp
  • J31007 (TetA C) - Amp
  • C0062 (LuxR w/o RBS) -Amp
  • J37033 (RBS/LuxR) from the 2007 distribution plate - Amp



Making plates
19 LB-agar-Ampi plates


Aleksandra
Overnight culture of 10ml LB + Kanamicin with the bacterial colony on the attc-t-attC-term plate from 07/15 (the colonies appeared only on the 07/19). We're not sure it's the transformation that worked, but we want to make sure.