IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/23

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<font color=red>Antoine</font>
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<br><u>Minipreps</u>
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<br><u>Nanodrop</u> to determine the amount of DNA in the miniprep samples:
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|Sample name || 260/280 ||260/230 ||ng/μL
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|  R0011 ||1.18 ||2.22||63
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| C0012 ||1.89||2.39 ||154.5
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| C0040 ||1.86 ||2.40||320.4
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| R0040 ||1.86||2.31||106.5
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| P1003 ||1.89 ||2.38||499.7
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<br><u>Glycerols</u>
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<br><u>Phage production</u>
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<br><u>Overnight culture </u>
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Revision as of 06:12, 2 August 2010

Memo cell Main project page
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Antoine


Minipreps


Nanodrop to determine the amount of DNA in the miniprep samples:

Sample name 260/280 260/230 ng/μL
R0011 1.18 2.2263
C0012 1.892.39 154.5
C0040 1.86 2.40320.4
R0040 1.862.31106.5
P1003 1.89 2.38499.7


Glycerols



Phage production



Overnight culture

Population counter Main project page
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Théotime
Restriction digest of the same minipreps as yestarday (concentration measured by nanodrop 07/22) using the same protocol as yesterday : 3µg of DNA per reaction.

  • RBS/LuxI (K081008) : 32µl DNA + 4µl Buffer 4 10x + 0.4µl BSA 100x + 2µl EcoRI-HF + 2µl SpeI
  • TetA(C) (J31007) in a triple volume : 60µl DNA + 7.5µl Buffer 3 10x + 0.8µl BSA 100x + 4µl XbaI + 4µl PstI
  • LacZ alpha (I732006) in a triple quantity : : 41µl DNA + 5.5µl Buffer 3 10x + 0.6µl BSA 100x + 4µl XbaI + 4µl PstI
  • LuxR (C0062), in triple : 59µl DNA + 7.5µl Buffer 3 10x + 0.7µl BSA 100x + 4µl XbaI + 4µl PstI
  • attC : 111µl DNA + 13µl Buffer 2 10x + 1.3µl BSA 100x + 2µl SpeI +2µl PstI
  • term/attC/mRFP : 91µl DNA + 11µl Buffer 3 10x + 1.1µl BSA 100x + 2µl XbaI + 2µl PstI
  • pLux (R0062) : 49µl DNA + 6µl Buffer 2 10x + 0.6µl BSA 100x + 2µl SpeI + 2µl PstI
  • RBS/GFP/term (E0240) : 26µl DNA + 3.5µl Buffer 3 10x + 0.4µl BSA 100x +2 µl XbaI + 2µl PastI


Stéphane
Transformation of TOP10 chemically competent E.coli with the overnight ligations from 07/22. Recuperation for 30 mins after the heat shock in SOC, then plating with 150µl of the transformmation on the ampicilin plate, incubation overnight at 37°C.

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



Théotime and Aleksandra
Gel electrophoresis of the restriction digest products, only for the future inserts.
Agarose gel 0.8%, 50V to separate the big inserts

  • TetA(C) (J31007) : 37µl + 7.4µl lb
  • TetA(C) (J31007) : 37µl + 7.4µl lb
  • ladder
  • LuxR (C0062) : 37µl + 7.4µl lb
  • LuxR (C0062) : 37µl + 7.4µl lb
  • -
  • term/attC/mRFP : 37µl + 7.4µl lb
  • term/attC/mRFP : 37µl + 7.4µl lb
  • term/attC/mRFP : 37µl + 7.4µl lb
  • -
  • RBS/GFP/term : 37µl + 7.4µl lb
  • ladder

Image:gel2307_1.jpgImage:gel2307_2.jpg


Agarose gel 3%, 50V to separate the small inserts

  • ladder (Quick load 100bp ladder), 5µl
  • LacZ alpha (I732006) : 27µl + 5.5µl lb
  • LacZ alpha (I732006) : 27µl + 5.5µl lb
  • -
  • RBS/LuxI (K081008) : 40µl + 8µl lb
  • RBS/LuxI (K081008) : 40µl + 8µl lb

Image:gel23072_1.jpg Image:gel23072_2.jpg


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


Stéphane
DNA purification from the 07/23 minipreps using the PCR purification kit.
Elution with 30µl water. In the result we have 2x28µl of each biobrick that will serve us as a vector for the ligation:

  • pLux (R0062)
  • attC


Raphaël
Minipreps : 4 minipreps of a 5ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL:

  • attC
  • term/attC/mRFP
  • attC-term/attC/mRFP (colony from the transformation plate of 07/21)
  • pLux-RBS/GFP/term (colony from the transformation plate of 07/21)


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

Sample name 260/280 260/230 ng/μL
attC(miniprep) 1.85 2.0966.6
t-attC-mRFP (miniprep) 1.852.17 78.3
pLux-GFP (miniprep) 1.86 2.38174.8
attC-t-attC-mRFP (miniprep) 1.832.2875.9
attC (PCR purif.) 1.70 1.3220.0
pLux (PCR purif.) 1.791.8870.3
LuxI (gel purif.) 1.71 0.055.1
LacZ (gel purif.) 2.26 0.02 3.1
TetA(C) (gel purif.) 1.92 0.08 10.8
GFP (gel purif.) 1.80 0.08 6.2
t-attC-mRFP (gel purif.) 1.400.01 1.6
LuxR (gel purif.) 1.66 0.06 3.4


Obviously, there is not enough DNA after gel purification to do a ligation...


Overnight culture
2x20mL per biobrick, the goal is to do minipreps on 10mL of pellet tomorrow (increase the initial amount of DNA >> increase the amount of DNA after gel purification !)

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


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