Team:Paris Saclay/Notebook/August/9

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Nanodrop
Nanodrop
* Bba_K1155007 in clone 10 : 38ng/µl   
* Bba_K1155007 in clone 10 : 38ng/µl   
-
* Bba_K1155007 in clone 14: 48.5ng/µl  
+
* Bba_K1155007 in clone 14 : 48.5ng/µl  
-
* Bba_K1155007 in clone 15: 52 ng/µl   
+
* Bba_K1155007 in clone 15 : 52 ng/µl   
{|
{|
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====Objective : Obtaining FNR and BphR2 proteins====
====Objective : Obtaining FNR and BphR2 proteins====
-
(((((((((===='''1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I'''====
+
===='''1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification'''====
{|
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| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
-
*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
+
*Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye
-
*Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
+
*Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye
-
*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
+
*Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
-
*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
+
*Well 5 : 5µL of FNR Part I+1µl of 6X loading dye
-
*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
+
*Well 6 : 5µL of FRN Part II+1µl of 6X loading dye
-
*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
+
*Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye
*Gel : 0.8%
*Gel : 0.8%
|}
|}
-
we have no fragment so we must do again these PCRs )))))))))))))))))
+
Expected size :
 +
* BphR2 Part I :
 +
* BphR2 Part II :
 +
* RBS-BphR2 Part I :
 +
* FNR Part I :
 +
* FNR Part II :
 +
* RBS-FNR PartI :
 +
 
 +
{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost all our PCR fragments. We will do the PCR again.
 +
|}
===='''2 - PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I'''====
===='''2 - PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I'''====

Revision as of 20:12, 16 September 2013

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Contents

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

((((((((((((((((((((====1 - Obtaining Δ fnr E. coli strain by transduction to test our biobricks====

Abdou, Anais, Damir, Nadia, XiaoJing

We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain was too old ( 2001)

Protocol : transduction

Ps908transduction.jpg

Picture: lysed cells comparison.

  • 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
  • 50µl phage: the petri dish is clear, bacteria are lysed by phages.
  • We used a wild type strain to keep a stock and a Δ fnr E. coli strain to obtain phages that might have encapsidated a Δfnr::Km fragment.

10µl,50µl and 100µl petri dishes are clear so phages are multiplied.

We let the antibiotic over night to select the right strain. ))))))))))))))))))))))


Objective : obtaining Bba_K1155007

1 - Extraction of Bba_K115007 from DH5α

Abdou

Protocol : Hight copy plamid extraction

We used colonies number 10, 14 and 15.

Nanodrop

  • Bba_K1155007 in clone 10 : 38ng/µl
  • Bba_K1155007 in clone 14 : 48.5ng/µl
  • Bba_K1155007 in clone 15 : 52 ng/µl

CONCLUSION !!!!!!!! We will sequence our plasmid.


A - Aerobic/Anaerobic regulation system / B - PCB sensing system

Objective : Obtaining FNR and BphR2 proteins

1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye
  • Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye
  • Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
  • Well 5 : 5µL of FNR Part I+1µl of 6X loading dye
  • Well 6 : 5µL of FRN Part II+1µl of 6X loading dye
  • Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye
  • Gel : 0.8%

Expected size :

  • BphR2 Part I :
  • BphR2 Part II :
  • RBS-BphR2 Part I :
  • FNR Part I :
  • FNR Part II :
  • RBS-FNR PartI :

We lost all our PCR fragments. We will do the PCR again.

2 - PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I

Anaïs, Damir, Nadia, XiaoJing

Used quantities :

  • Bphr2 Part I :
    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer Phusion : 10µL
    • DNA of Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • Bphr2 Part II :
    • Oligo 56F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-Bphr2 Part I :
    • Oligo 58F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part I :
    • Oligo 59F : 1µL
    • Oligo 60R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part II :
    • Oligo 61F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-FNR Part I :
    • Oligo 63F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL

PCR Program :

...

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