Team:Paris/17 August 2009

From 2009.igem.org

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==NoteBook==
==NoteBook==
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{{Paris2009_Calendar}}
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{| style="color:#1b2c8a;background-color:#0;" cellpadding="0" cellspacing="1" border="0" bordercolor="#fff" width="100%" align="left"
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{{Paris2009_Calendar_Link|16_August_2009|18_August_2009}}
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!align="center"|{{ #calendar: title=Team:Paris |year=2009 | month=06 }}
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<center> '''August 17th''' </center>
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!align="center"|{{ #calendar: title=Team:Paris |year=2009 | month=07 }}
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!align="center"|{{ #calendar: title=Team:Paris |year=2009 | month=08 }}
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!align="center"|{{ #calendar: title=Team:Paris |year=2009 | month=09 }}
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!align="center"|{{ #calendar: title=Team:Paris |year=2009 | month=10 }}
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|}
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<html>
<html>
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</html>
</html>
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==Lab work==
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==Brain work==
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<div class="romain">
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Competent Bacteria
 +
</div>
 +
<div class="experience">
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*Competent bacteria
 +
**1/50 ON culture of New DH5&alpha;
 +
**OD<sub>600</sub>=0,6
 +
*Transformation with puc19 (10pg/µl)
 +
**250µl of  competent DH5&alpha;
 +
**1µl puc19
 +
**Leave 20 min in ice
 +
**45s at 42°C
 +
**Leave 2 min in ice
 +
**Add 1ml of hot LB (42°C)
 +
**1h under agitation
 +
**Put on plate (LB agar + Amp)
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edit please ^^
 
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<html>
 
</div>
</div>
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<div id="paris_content_boxtop">
 
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</div>
 
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<div id="paris_content">
 
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</html>
 
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==Lab work==
 
<div class="charlotte">
<div class="charlotte">
Vector Dephosphorylation
Vector Dephosphorylation
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<div class="experience">
<div class="experience">
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Dephosphorylation:
 
pSB1A3 digested by '''EcoRI/PstI''', '''XbaI/PstI''' and '''EcoRI/SpeI''' have to be dephosphorylated before the ligation process.
pSB1A3 digested by '''EcoRI/PstI''', '''XbaI/PstI''' and '''EcoRI/SpeI''' have to be dephosphorylated before the ligation process.
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Mix :  
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''Mix'' :
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Vector : 30uL
Vector : 30uL
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Buffer : 3,5 uL
Buffer : 3,5 uL
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 +
''Protocol''
Put the solution at 37°C for 30 min
Put the solution at 37°C for 30 min
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[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D13 D13] (X/P): 0,94 ug/mL
[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D13 D13] (X/P): 0,94 ug/mL
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[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D14 D14]: 1,2 ug/ml
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[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D14 D14] (X/P): 1,2 ug/ml
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pSB1A3 (X/P): 2,1 ug/ml
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[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#P16 pSB1A3] (X/P): 2,1 ug/ml
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'''Mix''' : total volume = 10 uL
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''Mix : total volume = 10 uL''
vector (pSB1A3) : 2 uL
vector (pSB1A3) : 2 uL
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<div class="experience">
<div class="experience">
-
Transformation of BBa_B0034 (AmpR), BBa_E0030(KanR), pSB3C5 (CmR), pINTE3 (Col E3, AmpR), pTA (pTet-Col A, AmpR), pINTg3p (g3p Term, AmpR), pINK2(TolRII, AmpR) and previous ligation using the "heat choc" protocol
+
'''''Transformation of (using the "heat choc" protocol)''''' :
 +
 
 +
- BBa_B0034 (''AmpR'')
 +
 
 +
- BBa_E0030(''KanR'')
 +
 
 +
- pSB3C5 (''CmR'')
 +
 
 +
- pINTE3 (Col E3, ''AmpR'')
 +
 
 +
- pTA (pTet-Col A, ''AmpR'')  
 +
 
 +
- pINTg3p (g3p Term, ''AmpR'')
 +
 
 +
- pINK2(TolRII, ''AmpR'')
 +
 
 +
- Ligation 1 ( set up on Friday 14th of august w/ [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D11 D11 (RBS-Tet digested by Xba/Pst)] and vecteur [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D12 D12 (pSB2K3 w/ pLac digested by Spe/Pst)] (''KanR'')
 +
 
 +
- previous ligation (w/ D13 and D14) (''AmpR'')
</div>
</div>
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==To do list==
+
<div class="vicard">
 +
Transformation Check/PCR on colonies
 +
</div>
 +
<div class="experience">
 +
<strong>Transformation check:</strong>
 +
 
 +
The x10mix ligation after centrifugation and put at 16°C overnight had work.
 +
 
 +
using less incubation time at RT seems to work better.
 +
 
 +
using less insert seems to work better
 +
 
 +
<strong>PCR on colonies:</strong>
 +
 
 +
using for 1 tube:
 +
 
 +
Master Mix 2X : 12,5µL
 +
 
 +
Oligo VF2 (10µM): 0,5µL
 +
 
 +
Oligo VR  (10µM): 0,5µL
 +
 
 +
bacteria in H20 (see PCR on colonies protocol): 5µL
 +
 
 +
H20 : 6,5µL
 +
 
 +
'''PCR on colonies Check OK for colonie 3'''
 +
<center>[[Image:3e_colobis.JPG]]</center>
 +
 
 +
 
 +
</div>
 +
 
 +
 
-
edit please ^^
+
{{Paris2009_Calendar_Link|16_August_2009|18_August_2009}}

Latest revision as of 09:38, 7 September 2009

NoteBook

June
MTWTFSS
[http://2009.igem.org/Team:Paris/1_June_2009 1] [http://2009.igem.org/wiki/index.php?title=Team:Paris/2_June_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Paris/3_June_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Paris/4_June_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Paris/5_June_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Paris/6_June_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Paris/7_June_2009&action=edit 7]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/8_June_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Paris/9_June_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Paris/10_June_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Paris/11_June_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Paris/12_June_2009&action=edit 12] [http://2009.igem.org/Team:Paris/13_June_2009 13] [http://2009.igem.org/wiki/index.php?title=Team:Paris/14_June_2009&action=edit 14]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/15_June_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Paris/16_June_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Paris/17_June_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Paris/18_June_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Paris/19_June_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Paris/20_June_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Paris/21_June_2009&action=edit 21]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/22_June_2009&action=edit 22] [http://2009.igem.org/Team:Paris/23_June_2009 23] [http://2009.igem.org/wiki/index.php?title=Team:Paris/24_June_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Paris/25_June_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Paris/26_June_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Paris/27_June_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Paris/28_June_2009&action=edit 28]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/29_June_2009&action=edit 29] [http://2009.igem.org/Team:Paris/30_June_2009 30]
July
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Team:Paris/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Paris/2_July_2009&action=edit 2] [http://2009.igem.org/Team:Paris/3_July_2009 3] [http://2009.igem.org/wiki/index.php?title=Team:Paris/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Paris/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Paris/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Paris/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Paris/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Paris/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Paris/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Paris/12_July_2009&action=edit 12]
[http://2009.igem.org/Team:Paris/13_July_2009 13] [http://2009.igem.org/Team:Paris/14_July_2009 14] [http://2009.igem.org/Team:Paris/15_July_2009 15] [http://2009.igem.org/Team:Paris/16_July_2009 16] [http://2009.igem.org/Team:Paris/17_July_2009 17] [http://2009.igem.org/Team:Paris/18_July_2009 18] [http://2009.igem.org/Team:Paris/19_July_2009 19]
[http://2009.igem.org/Team:Paris/20_July_2009 20] [http://2009.igem.org/Team:Paris/21_July_2009 21] [http://2009.igem.org/Team:Paris/22_July_2009 22] [http://2009.igem.org/Team:Paris/23_July_2009 23] [http://2009.igem.org/Team:Paris/24_July_2009 24] [http://2009.igem.org/Team:Paris/25_July_2009 25] [http://2009.igem.org/Team:Paris/26_July_2009 26]
[http://2009.igem.org/Team:Paris/27_July_2009 27] [http://2009.igem.org/Team:Paris/28_July_2009 28] [http://2009.igem.org/Team:Paris/29_July_2009 29] [http://2009.igem.org/Team:Paris/30_July_2009 30] [http://2009.igem.org/Team:Paris/31_July_2009 31]
August
MTWTFSS
          [http://2009.igem.org/Team:Paris/1_August_2009 1] [http://2009.igem.org/Team:Paris/2_August_2009 2]
[http://2009.igem.org/Team:Paris/3_August_2009 3] [http://2009.igem.org/Team:Paris/4_August_2009 4] [http://2009.igem.org/Team:Paris/5_August_2009 5] [http://2009.igem.org/Team:Paris/6_August_2009 6] [http://2009.igem.org/Team:Paris/7_August_2009 7] [http://2009.igem.org/Team:Paris/8_August_2009 8] [http://2009.igem.org/Team:Paris/9_August_2009 9]
[http://2009.igem.org/Team:Paris/10_August_2009 10] [http://2009.igem.org/Team:Paris/11_August_2009 11] [http://2009.igem.org/Team:Paris/12_August_2009 12] [http://2009.igem.org/Team:Paris/13_August_2009 13] [http://2009.igem.org/Team:Paris/14_August_2009 14] [http://2009.igem.org/Team:Paris/15_August_2009 15] [http://2009.igem.org/Team:Paris/16_August_2009 16]
[http://2009.igem.org/Team:Paris/17_August_2009 17] [http://2009.igem.org/Team:Paris/18_August_2009 18] [http://2009.igem.org/Team:Paris/19_August_2009 19] [http://2009.igem.org/Team:Paris/20_August_2009 20] [http://2009.igem.org/Team:Paris/21_August_2009 21] [http://2009.igem.org/Team:Paris/22_August_2009 22] [http://2009.igem.org/Team:Paris/23_August_2009 23]
[http://2009.igem.org/Team:Paris/24_August_2009 24] [http://2009.igem.org/Team:Paris/25_August_2009 25] [http://2009.igem.org/Team:Paris/26_August_2009 26] [http://2009.igem.org/Team:Paris/27_August_2009 27] [http://2009.igem.org/Team:Paris/28_August_2009 28] [http://2009.igem.org/Team:Paris/29_August_2009 29] [http://2009.igem.org/Team:Paris/30_August_2009 30]
[http://2009.igem.org/Team:Paris/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/Team:Paris/1_September_2009 1] [http://2009.igem.org/Team:Paris/2_September_2009 2] [http://2009.igem.org/Team:Paris/3_September_2009 3] [http://2009.igem.org/Team:Paris/4_September_2009 4] [http://2009.igem.org/Team:Paris/5_September_2009 5] [http://2009.igem.org/Team:Paris/6_September_2009 6]
[http://2009.igem.org/Team:Paris/7_September_2009 7] [http://2009.igem.org/Team:Paris/8_September_2009 8] [http://2009.igem.org/Team:Paris/9_September_2009 9] [http://2009.igem.org/Team:Paris/10_September_2009 10] [http://2009.igem.org/Team:Paris/11_September_2009 11] [http://2009.igem.org/Team:Paris/12_September_2009 12] [http://2009.igem.org/Team:Paris/13_September_2009 13]
[http://2009.igem.org/Team:Paris/14_September_2009 14] [http://2009.igem.org/Team:Paris/15_September_2009 15] [http://2009.igem.org/Team:Paris/16_September_2009 16] [http://2009.igem.org/Team:Paris/17_September_2009 17] [http://2009.igem.org/Team:Paris/18_September_2009 18] [http://2009.igem.org/Team:Paris/19_September_2009 19] [http://2009.igem.org/Team:Paris/20_September_2009 20]
[http://2009.igem.org/Team:Paris/21_September_2009 21] [http://2009.igem.org/Team:Paris/22_September_2009 22] [http://2009.igem.org/Team:Paris/23_September_2009 23] [http://2009.igem.org/Team:Paris/24_September_2009 24] [http://2009.igem.org/Team:Paris/25_September_2009 25] [http://2009.igem.org/Team:Paris/26_September_2009 26] [http://2009.igem.org/Team:Paris/27_September_2009 27]
[http://2009.igem.org/Team:Paris/28_September_2009 28] [http://2009.igem.org/Team:Paris/29_September_2009 29] [http://2009.igem.org/Team:Paris/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/Team:Paris/1_October_2009 1] [http://2009.igem.org/Team:Paris/2_October_2009 2] [http://2009.igem.org/Team:Paris/3_October_2009 3] [http://2009.igem.org/Team:Paris/4_October_2009 4]
[http://2009.igem.org/Team:Paris/5_October_2009 5] [http://2009.igem.org/Team:Paris/6_October_2009 6] [http://2009.igem.org/Team:Paris/7_October_2009 7] [http://2009.igem.org/Team:Paris/8_October_2009 8] [http://2009.igem.org/Team:Paris/9_October_2009 9] [http://2009.igem.org/Team:Paris/10_October_2009 10] [http://2009.igem.org/Team:Paris/11_October_2009 11]
[http://2009.igem.org/Team:Paris/12_October_2009 12] [http://2009.igem.org/Team:Paris/13_October_2009 13] [http://2009.igem.org/Team:Paris/14_October_2009 14] [http://2009.igem.org/Team:Paris/15_October_2009 15] [http://2009.igem.org/Team:Paris/16_October_2009 16] [http://2009.igem.org/Team:Paris/17_October_2009 17] [http://2009.igem.org/Team:Paris/18_October_2009 18]
[http://2009.igem.org/Team:Paris/19_October_2009 19] [http://2009.igem.org/Team:Paris/20_October_2009 20] [http://2009.igem.org/Team:Paris/21_October_2009 21] [http://2009.igem.org/Team:Paris/22_October_2009 22] [http://2009.igem.org/Team:Paris/23_October_2009 23] [http://2009.igem.org/Team:Paris/24_October_2009 24] [http://2009.igem.org/Team:Paris/25_October_2009 25]
[http://2009.igem.org/Team:Paris/26_October_2009 26] [http://2009.igem.org/Team:Paris/27_October_2009 27] [http://2009.igem.org/Team:Paris/28_October_2009 28] [http://2009.igem.org/Team:Paris/29_October_2009 29] [http://2009.igem.org/Team:Paris/30_October_2009 30] [http://2009.igem.org/Team:Paris/31_October_2009 31]

← Yesterday - Tomorrow →

August 17th

Lab work

Competent Bacteria

  • Competent bacteria
    • 1/50 ON culture of New DH5α
    • OD600=0,6
  • Transformation with puc19 (10pg/µl)
    • 250µl of competent DH5α
    • 1µl puc19
    • Leave 20 min in ice
    • 45s at 42°C
    • Leave 2 min in ice
    • Add 1ml of hot LB (42°C)
    • 1h under agitation
    • Put on plate (LB agar + Amp)

Vector Dephosphorylation


pSB1A3 digested by EcoRI/PstI, XbaI/PstI and EcoRI/SpeI have to be dephosphorylated before the ligation process.


Mix :

Vector : 30uL

Antarctic Phosphatase : 2uL

Buffer : 3,5 uL


Protocol

Put the solution at 37°C for 30 min

Add 2 uL of enzyme

37°C for 30 min

65°C for 20 min for heat inactivation


Ligation


D13 (X/P): 0,94 ug/mL

D14 (X/P): 1,2 ug/ml

pSB1A3 (X/P): 2,1 ug/ml


Ligation (2hrs at RT) of pSB1A3 w/ D13 (ompA signal) or D14 (TolRII)


Mix : total volume = 10 uL

vector (pSB1A3) : 2 uL

insert (A11 or A13) : 4uL

10X T4DNA Ligase buffer : 1 uL

T4 DNA Ligase : 0,5 uL

H2O: 2,5 uL


Negatif control : same mix as the previous one but without the insert. In these conditions the amount of water was increased until 6,5 uL.


Transformation

Transformation of (using the "heat choc" protocol) :

- BBa_B0034 (AmpR)

- BBa_E0030(KanR)

- pSB3C5 (CmR)

- pINTE3 (Col E3, AmpR)

- pTA (pTet-Col A, AmpR)

- pINTg3p (g3p Term, AmpR)

- pINK2(TolRII, AmpR)

- Ligation 1 ( set up on Friday 14th of august w/ D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst) (KanR)

- previous ligation (w/ D13 and D14) (AmpR)

Transformation Check/PCR on colonies

Transformation check:

The x10mix ligation after centrifugation and put at 16°C overnight had work.

using less incubation time at RT seems to work better.

using less insert seems to work better

PCR on colonies:

using for 1 tube:

Master Mix 2X : 12,5µL

Oligo VF2 (10µM): 0,5µL

Oligo VR (10µM): 0,5µL

bacteria in H20 (see PCR on colonies protocol): 5µL

H20 : 6,5µL

PCR on colonies Check OK for colonie 3

3e colobis.JPG



← Yesterday - Tomorrow →