Team:Paris/15 August 2009

From 2009.igem.org

(Difference between revisions)
(→Lab work)
(→Lab work)
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==Lab work==
==Lab work==
-
 
+
<div class="guillaume">
 +
Transformation
 +
</div>
 +
<div class="experience">
 +
*For overnight (at 16°C) ligation solution of g3p in pSB1A3
 +
*For overnight (at 16°C) ligation solution of ClyA in pSB1A3
 +
</div>
 +
<div class="guillaume">
 +
Miniprep
 +
</div>
 +
<div class="experience">
 +
*ptet
 +
*pBad/araC
 +
*Double terminater
 +
</div>
 +
<div class="guillaume">
 +
Glycerol Stock
 +
</div>
 +
<div class="experience">
 +
*New strains
 +
**[S35] AA93 &Delta;fecA
 +
**[S36] fecIR mutant
 +
**[S37] fecIR wt
 +
**[S38] fecA
 +
</div>
 +
<div class="guillaume">
 +
Yesterday transformations results
 +
</div>
 +
<div class="experience">
 +
*tatABC
 +
**No bacteria
 +
*pFecA
 +
**Negative control = 0 bacteria
 +
**1:1 raport = 43 bacteria
 +
**1:7 raport = 2 bacteria
 +
**1:7 concentred = Too much
 +
*g3p
 +
**Negative control = 0 bacteria
 +
**1:1 raport = 1 bacteria
 +
**1:7 raport = 0 bacteria
 +
**1:7 concentred = 16 bacteria
 +
</div>
 +
<div class="guillaume">
 +
PCR screening protocol
 +
</div>
 +
<div class="experience">
 +
*Program named COLONIES, use with VF2 and VR oligos
 +
**Lid 105°C
 +
**1: 99°C 2min
 +
**2: Sound
 +
**3: Pause until press "Enter" button
 +
**4: 95°C 5min
 +
**5: 95°C 30sec
 +
**6: 55°C 30sec
 +
**7: 72°C [45sec] (1kb/bp, caution the universal oligo add 156bp)
 +
**8: Goto 5 29x
 +
**9:72°C 7min
 +
**10: Hold at 4°C
 +
*Preparation of the ON culture (for Glycerol stock and Miniprep of the positives clones)
 +
**7ml of LB+ Amp (because we use pSB1A3 as vector)
 +
**Bacterial solution
 +
**100uL of H2O and few bacteria in a PCR tube
 +
**Put the cone in LB solution for ON culture
 +
**Put the PR tube in PCR and run the fist part of the program
 +
*PCR mix
 +
**Master Mix (2x) : 12,5ul
 +
**Oligo VF2 (10uM) : 0,5ul
 +
**Oligo VR (10uM) : 0,5ul
 +
**H2O : 6,5ul
 +
**Bacteria hoted at 99°C by PCR program : 5ul
 +
*Put PCR tube withe the PCR mix in the PCR and run the second part of the program (just push the "Enter" button)
 +
</div>
 +
<div class="guillaume">
 +
PCR screening
 +
</div>
 +
<div class="experience">
 +
*For 6 clones
 +
*Negative control without bacteria
 +
*"Positive" control with pSB1A3 wich have the mRFP (1050bp)
 +
</div>
 +
<div class="guillaume">
 +
Gel migration of the PCR screening
 +
</div>
 +
<div class="experience">
 +
*All the clone have se same profil (more than 300bp and under 400bp) and it seem to be good...Confirmation tomorow by enzymes digestion.
 +
*Negative control is...negative
 +
*Positive control is...positif but have a second band. (Must be confirmed)
[[Image:150809 Colony PCR pFecA.png]]
[[Image:150809 Colony PCR pFecA.png]]
-
 
+
</div>
-
 
+
<html>
<html>
</div>
</div>

Revision as of 22:09, 16 August 2009

Contents

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]


Brain work

edit please ^^



Lab work

Transformation

  • For overnight (at 16°C) ligation solution of g3p in pSB1A3
  • For overnight (at 16°C) ligation solution of ClyA in pSB1A3

Miniprep

  • ptet
  • pBad/araC
  • Double terminater

Glycerol Stock

  • New strains
    • [S35] AA93 ΔfecA
    • [S36] fecIR mutant
    • [S37] fecIR wt
    • [S38] fecA

Yesterday transformations results

  • tatABC
    • No bacteria
  • pFecA
    • Negative control = 0 bacteria
    • 1:1 raport = 43 bacteria
    • 1:7 raport = 2 bacteria
    • 1:7 concentred = Too much
  • g3p
    • Negative control = 0 bacteria
    • 1:1 raport = 1 bacteria
    • 1:7 raport = 0 bacteria
    • 1:7 concentred = 16 bacteria

PCR screening protocol

  • Program named COLONIES, use with VF2 and VR oligos
    • Lid 105°C
    • 1: 99°C 2min
    • 2: Sound
    • 3: Pause until press "Enter" button
    • 4: 95°C 5min
    • 5: 95°C 30sec
    • 6: 55°C 30sec
    • 7: 72°C [45sec] (1kb/bp, caution the universal oligo add 156bp)
    • 8: Goto 5 29x
    • 9:72°C 7min
    • 10: Hold at 4°C
  • Preparation of the ON culture (for Glycerol stock and Miniprep of the positives clones)
    • 7ml of LB+ Amp (because we use pSB1A3 as vector)
    • Bacterial solution
    • 100uL of H2O and few bacteria in a PCR tube
    • Put the cone in LB solution for ON culture
    • Put the PR tube in PCR and run the fist part of the program
  • PCR mix
    • Master Mix (2x) : 12,5ul
    • Oligo VF2 (10uM) : 0,5ul
    • Oligo VR (10uM) : 0,5ul
    • H2O : 6,5ul
    • Bacteria hoted at 99°C by PCR program : 5ul
  • Put PCR tube withe the PCR mix in the PCR and run the second part of the program (just push the "Enter" button)

PCR screening

  • For 6 clones
  • Negative control without bacteria
  • "Positive" control with pSB1A3 wich have the mRFP (1050bp)

Gel migration of the PCR screening

  • All the clone have se same profil (more than 300bp and under 400bp) and it seem to be good...Confirmation tomorow by enzymes digestion.
  • Negative control is...negative
  • Positive control is...positif but have a second band. (Must be confirmed)

150809 Colony PCR pFecA.png

To do list

edit please ^^

Retrieved from "http://2009.igem.org/Team:Paris/15_August_2009"