IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/27: Difference between revisions

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<br><u>PCR purification</u>
<br><u>PCR purification</u>
*P1003
*P1003 14.8 ng/uL
*S173
*S173 17.8 ng/uL
I have made a PCR purification because the fragment cut beetween S and P restriction site will be remove by the column.
I have made a PCR purification because the fragment cut beetween S and P restriction site will be remove by the column.


<br><u>Electrophoresis</u>
<br><u>Electrophoresis</u>
<br><u>Gel extraction</u>
*C0012 11.2 ng/uL
*J23110 well 1 12.4 ng/uL
*J23110 well 2 30.2 ng/uL
<br><u>Overnigth culture</u>




Revision as of 08:43, 2 August 2010

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Miniprep

  • S200 (Constitutif promoter + standard RBS + tetR) Vf=60 uL 72.9 ng/ul
  • S173 (standard RBS + tetR) Vf=60 uL 208.3 ng/uL


Restriction digest Vf=20 uL

Sample name DNA (1ug) enzyme Buffer H2O BSA
S173 4.8 1 S/P 2(B2) 11 0.2
J23110 (Vf=40uL) 2 2 S/P 4(B2) 29.6 0.4
B0015 7.4 2 X/P 4 (B2) 24.2 0.4
P1003 2 1 S/P 2 (B2) 13.8 0.2
C0012 6.5 1 S/P 2 (B2) 9.3 0.2


PCR purification

  • P1003 14.8 ng/uL
  • S173 17.8 ng/uL

I have made a PCR purification because the fragment cut beetween S and P restriction site will be remove by the column.


Electrophoresis


Gel extraction

  • C0012 11.2 ng/uL
  • J23110 well 1 12.4 ng/uL
  • J23110 well 2 30.2 ng/uL


Overnigth culture


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Aleksandra and Raphaël
Transformation of TOP10 cells (25µl/transformation) with the 07/26 ligations:

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


Incubation at 37°C for 21h.


Aleksandra
Miniprep
4x10ml cultures of term/attC/mRFP, elution with 30µl EB from each column = 120µl DNA in total


Nanodrop quantification of DNA in the samples

Sample name 260/280 260/230 ng/µl
term/attC/mRFP 1.99 2.04 97.8
term/attC/mRFP, concentrated 1.82 2.01 114.8


We concentrated 60µl of the sample using the PCR purification kit, eluting in 30µl warm water, but the concentration wasn't successful enough. Anyway, we used the concentrated sample for the restriction digest.


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

  • 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
  • 40µl attC (miniprep of 07/25) + 1µl SpeI + 1µl PstI + 4.7µl buffer3 10x + 0.45µl BSA 100x


Théotime
DNA purification from the restriction digest reaction using the PCR purification kit

  • attC eluted with 30µl of water


Making Amp plates
21 plates

Raphaël
Gel electrophoresis (1.5% agarose, 50V)

  • ladder 1kb
  • -
  • term/attC/mRFP
  • -
  • -
  • -


Gel purification and nanodrop to quantify DNA

Sample name 260/280 260/230 ng/µl
attC (PCR purif.kit) 1.67 1.01 6.2
term/attC/mRFP (gel purif.) 2.01 0.05 14.4


We often have very low 260/230 values : probably contamination with the solvents.


Ethanol precipitation of the term/attC/mRFP (gel purification) to concentrate the sample.
Following this protocol : [1]
60µl ethanol + 2.4µl sodium acetate 3M + 24µl term/attC/mRFP, incubation at -20°C overnight.


Aleksandra and Raphaël


Overnight cultures
10mL LB

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



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