IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/27

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*S173 (standard RBS + tetR) Vf=60 uL 208.3 ng/uL
*S173 (standard RBS + tetR) Vf=60 uL 208.3 ng/uL
-
<br><u>Miniprep</u>  
+
<br><u>Restriction digest</u> 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
 +
|}
 +
 
 +
<br><u>PCR purification</u>
 +
*P1003
 +
*S173
 +
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>
 +
 
|-
|-

Revision as of 11:34, 2 August 2010

Memo cell Main project page
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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 nameDNA (1ug)enzymeBufferH2OBSA
S173 4.8 1 S/P 2(B2) 11 0.2
J23110 (Vf=40uL) 2 2 S/P 4(B2) 29.60.4
B0015 7.42 X/P 4 (B2)24.20.4
P1003 21 S/P 2 (B2)13.80.2
C0012 6.51 S/P 2 (B2)9.30.2


PCR purification

  • P1003
  • S173

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


Electrophoresis


Population counter Main project page
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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 260/280 260/230ng/µl
term/attC/mRFP1.99 2.0497.8
term/attC/mRFP, concentrated 1.822.01 114.8


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
  • -
  • -
  • -

Image:gel2707.jpg


Gel purification and nanodrop to quantify DNA

Sample name260/280260/230ng/µl
attC (PCR purif.kit) 1.67 1.016.2
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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