Team:Paris Saclay/Notebook/August/14

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Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining ...

1 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI/SpeI

Nadia

  • Buffer FD : 2µL
  • H2O : 6µL
  • DNA : 10µL
  • SpeI FD : 1µL
  • EcoRI FD : 1µL

2 - Electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI/SpeI

Anaïs, Nadia

PSgel2 1408.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • PSB1C3 : 2070kb
  • NarK, NarG, NirB : 200kb

We obtained NarK, NarG, NirB fragments at the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes AND ADN ?!??????????

3 - Digestion of Bba_K1155000, Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

  • Digestion by SpeI :
    • Buffer : 2µL
    • SpeI : 2µL
    • ADN : 15µL
    • H20 : 1µL
  • Digestion by EcoRI and SpeI :
    • Buffer : 3µL
    • SpeI : 2µL
    • EcoRI : 2µL
    • ADN : 20µL
    • H20 : 3µL

4 - Electrophoresis to check the digestion of Bba_K1155000, Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

PSgel3 1408.jpg
  • Well 1 : 5µL of BBa_K1155000 digested by SpeI+1µl of 6X loading dye
  • Well 2 : 5µL of BBa_K1155006 digested by SpeI+1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155005 digested by SpeI+1µl of 6X loading dye
  • Well 4 : 5µL of BBa_K1155004 digested by SpeI+1µl of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of BBa_K1155000 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 7 : 5µL of BBa_K1155006 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 8 : 5µL of BBa_K1155005 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 9 : 5µL of BBa_K1155004 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • Pfnr in PSB1C3 : ...
  • Nar K in PSB1C3, NarG in PSB1C3, NirB in PSB1C3 : ...
  • PSB1C3 : 2070kb
  • NarK, NarG, NirB : 200kb
  • Pfnr : ...

Well 1 : we have done one digestion so we have to obtain one fragment, we have two stripes, the digestion of Bba_K1155000 by SpeI wasn't good. Well 2, 3, 4, 6, 7 : we obtain NarG, NarK and NirB in PSB1C3 and Pfnr, NarK fragments at the right size, we can purify it. Well 8, 9 : we have done two digestions so we have to obtain two fragments, we have only one stripe, the digestion of Bba_K1155005 and Bba_K1155004 by EcoRI/SpeI wasn't good.

5 - Gel purification of the digestion of Bba_K1155000 and Bba_K1155006 by EcoRI/SpeI and Bba_K1155004, Bba_K1155005 and Bba_K1155006 by SpeI

Anaïs, Nadia

Protocol : Gel purification

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Picture : Electrophoresis gel of the digestion of Bba_K1155000, Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI

  • The cutting for the gel purification was good.

Nanodrop :

  • Pfnr : 13.7ng/µL
  • NarK : 27.9ng/µL
  • NarK in PSB1C3 : 87.1ng/µL
  • NarG in PSB1C3 : 39.2ng/µL
  • NirB in PSB1C3 : 37.7ng/µL

CONCLUSION

6 - Gel purification of the digestion of Bba_K1155003 and Bba_K1155007 by XBaI/PstI

Nadia

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Picture : Electrophoresis gel of the digestion of Bba_K1155003 and Bba_K1155007 by XBaI/PstI

  • Thanks to electrophoresis we separated all the quantity of our digestion.

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

Objective : obtaining FNR and BphR2 proteins (Gibson assembly)

1 - Gibson assembly

PRECISIONS !!!!!!!!!!!!!!!!!!!!!!

2 - Electrophoresis of PCR products : BphR2 Part I

Damir

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  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I+1µL of 6X loading dye
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  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I+1µL of 6X loading dye
  • Well 3 : 5µL of BphR2 Part I+1µL of 6X loading dye

Expected sizes :

  • BphR2 Part I : 178kb

On the first gel, all deposits disappear so we did the electrophoresis again. On the second gel, we didn't obtain stripes at the good size. We do the PCR again using new quantities and a new PCR program.

3 - PCR of BphR2 Part I

Damir

Used quantities :

  • Oligo 54F : 2µL
  • Oligo 55R : 2µL
  • DNA : 1µL
  • Buffer Phusion : 10µL
  • dNTP : 1µL
  • Phusion : 1µL
  • DMS9 : 2µL ??????????????????????????????????
  • H2O : 31µL

PCR program :

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