Team:Paris Saclay/Notebook/August/12

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(Difference between revisions)
Line 16: Line 16:
Used quantities :  
Used quantities :  
-
* Bba_K1155000 :  
+
* Pfnr in PSB1C3 (Bba_K1155000) :  
** Buffer : 5µL
** Buffer : 5µL
** H2O : 38µL
** H2O : 38µL
Line 23: Line 23:
** PstI : 1µL
** PstI : 1µL
-
* Bba_K1155007 :  
+
* RBS-LacZ-Term in PSB1C3 (Bba_K1155007) :  
** Buffer : 5µL  
** Buffer : 5µL  
** H2O : 23µL
** H2O : 23µL
Line 30: Line 30:
** PstI : 1µL
** PstI : 1µL
-
* Bba_K1155003 :  
+
* RBS-AmilCP-Term in PSB1C3 (Bba_K1155003) :  
** Buffer : 5µL
** Buffer : 5µL
** H2O : 33µL
** H2O : 33µL

Revision as of 16:09, 12 August 2013

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Contents

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining Pfnr_RBS-LacZ-Term in PSB1C3

1 - Digestion of Bba_K1155000, Bba_K1155007 and Bba_K1155003

Anaïs, Nadia, XiaoJing

Protocol : digestion

Used quantities :

  • Pfnr in PSB1C3 (Bba_K1155000) :
    • Buffer : 5µL
    • H2O : 38µL
    • Pfnr : 5µL
    • SpeI : 1µL
    • PstI : 1µL
  • RBS-LacZ-Term in PSB1C3 (Bba_K1155007) :
    • Buffer : 5µL
    • H2O : 23µL
    • RBS-LacZ-Term : 20µL
    • XBal : 1µL
    • PstI : 1µL
  • RBS-AmilCP-Term in PSB1C3 (Bba_K1155003) :
    • Buffer : 5µL
    • H2O : 33µL
    • RBS-AmilCP-Term : 10µL
    • XBal : 1µL
    • PstI : 1µL


IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of digested Bba_K1155000+1µl of 6X loading dy
  • Well 3 : 5µL of digested Bba_K1155007+1µl of 6X loading dy
  • Well 4 : 5µL of digested Bba_K1155003+1µl of 6X loading dy
  • Gel : 0.8%

Expected sizes :

  • RBS_LacZ_Term : 3500 kb
  • PSB1C3 :

2 - ObtaµLining RBS_lacZ_ter.

Abdou

Clone 10,14 and 15 plamid extraction using nucleospin kit.

Protocol : hight copy plamid extraction

Results: concentration measured by nanodrop

  • clone 10: C=38ng/µl 260/280= 1.78
  • clone 14: C=48.5ng/µl 260/280=1.90
  • clone 15: C=52 ng/µl 260/280=1.78

We still have to sequence plasmids in order to verify our results.

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

1 - Gibson assembly.

August 1st PCR purification to be sure about the experiment. BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.

Protocol : PCR_clean_up

Results:

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
  • Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
  • Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
  • Well 5 : 5µL of FNR part1+1µl of 6X loading dy
  • Well 6 : 5µL of FRN part2+1µl of 6X loading dy
  • Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
  • Gel : 0.8%

we have no fragment so we must do again these PCRs

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