IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/28

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Antoine



Minipreps Vf=35 uL

  • B0015 115.4 ng/uL
  • B0015 200.4 ng/uL
  • S200 174 ng/uL


Restriction digest 2h 37°C follow by inactivation at 80°C 20 min Vf=20 uL

Sample nameDNA (1ug)enzymeBufferH2OBSA
B0015 (200.4 ng/uL) 5 1 X/P 2(B3) 10.8 0.2
B0015 5 1 X/P 2(B3) 10.8 0.2
B0015 5 1 X/P 2(B3) 10.8 0.2
B0015 5 1 X/P 2(B3) 10.8 0.2
S200 5.751 S/P 2 (B2)10.050.2
S200 5.751 S/P 2 (B2)10.050.2


Electrophoresis



Gel purification


Ligation follow by inactivation 65°C 10 min Vf=10uL

Vecteur (10ng)Insert(30ng)Buffer ligaseH2OT4 ligase
P1003(cut S+P) 0.7 BOO15(cut X+P) 2.1 1 2(B3) 5.70.5
C0012(cut S+P) 0.9 BOO15(cut X+P) 2.1 1 2(B3) 5.50.5
S173(cut S+P) 0.6 BOO15(cut X+P) 2.1 1 2(B3) 5.80.5
S200(cut S+P) 1.5 BOO15(cut X+P) 2.1 1 2(B3) 4.90.5


Transformation


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Raphaël
Ethanol precipitation of the term/attC/mRFP (gel purification) to concentrate the sample.
Following this protocol : [1]
Elution with 20µL of water


Aleksandra
Miniprep
10 mL of culture, following Eric's advices : skip the step with PB buffer, thoroughly wipe the tube and the column after wash with PE buffer, elution with 50µL of water at 55°C.

  • pSulib attC
  • pSulib term/attC/mRFP
  • TOP10 pSB1A2 pLux (R0062)
  • TOP10 pSB1A2 RBS/GFP/term (E0240)
  • TOP10 pSB1A2 pLux/GFP (1rst colony on the plate)
  • TOP10 pSB1A2 pLux/GFP (2nd colony on the plate)
  • TOP10 pSB1A2 pLux/GFP (3rd colony on the plate)
  • TOP10 pSB1A2 pLux/GFP (4th colony on the plate)


Nanodrop quantification of DNA amounts in our samples

Sample name260/280260/230ng/µl
attC 2.00 1.89 142.0
term/attC/mRFP 1.98 2.14 163.8
pLux 1.94 2.32 217.5
GFP 1.92 2.37 274.1
pLux/GFP 1 1.93 2.30 208.7
pLux/GFP 2 1.94 2.23 260.6
pLux/GFP 3 1.95 2.26 264.3
pLux/GFP 4 1.91 2.31 326.2
term/attC/mRFP (ethanol prec.) 1.27 0.12 5.2



Aleksandra and Raphaël
Restriction digest, incubation at 37°C for 2h.

  • 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
  • 20µl RBS/GFP/term + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
  • 20µl pLux + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl attC + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 5µl pLux/GFP 1 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 2 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 3 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 4 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water



Aleksandra and Théotime
Gel electrophoresis (1.5% agarose, 50V)

  • attC : 25µl + 5µl lb
  • Ladder 1Kb
  • term/attC/mRFP : 25µl + 5µl lb
  • -
  • pLux : 25µl + 5µl lb
  • -
  • GFP : 25µl + 5µl lb
  • -
  • pLux/GFP 1 : 20µl + 5µl lb
  • pLux/GFP 2 : 20µl + 5µl lb
  • pLux/GFP 3 : 20µl + 5µl lb
  • pLux/GFP 4 : 20µl + 5µl lb

Image:gel2807_1.jpg Image:gel2807_2.jpg


Gel purification with the Quiagen kit, elution in 50µl warm water.


Nanodrop quantification of DNA amounts in our samples

Sample name260/280260/230ng/µl
attC (gel purif.) 2.67 0.03 5.0*
term/attC/mRFP (gel purif.) 2.53 0.05 3.9*
pLux (gel purif.) 2.18 0.15 11.0
GFP (gel purif.) 1.84 0.2535.0


*These samples have no DNA : no peak at 260nm on the Nanodrop.
The yields of DNA are following : 8.8% for attC, 6% for term/attC/mRFP, 13% for pLux, 32% for RBS/GFP/term
Ligation
50ng of the vector and 150ng of insert in about 10μL of total volume. Ligation at 16°C overnight.

  • 15μL GFP (E0240) + 1.5μL pLux (R0062) + 2μL buffer 10x + 0.5μL T4 ligase + 1µl water


Overnight culture of the transformations from 07/27 (colonies on all the plates)
10mL LB + Amp, 2 colonies for each transformation

  • TetA(C) + stand.RBS
  • RBS/GFP/term + pLux
  • weakRBS/LuxR + Pmed
  • weakRBS/LuxR + Pstr
  • medRBS/LuxR + Pmed
  • medRBS/LuxR + Pstr
  • strRBS/LuxR + Pmed
  • strRBS/LuxR + Pstr
  • LacZ + weak RBS
  • LacZ + stand RBS
  • LuxI + term


Overnight culture from the glycerol stock
10mL LB

  • 2x attC
  • 2x term/attC/mRFP



Overnight culture of the transformation from 07/21, 5 colonies

  • pLux/GFP/term


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