Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining ...

1 - Electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI

Anaïs, Nadia

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 :

• PSB1C33 : 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 ?!

2 - 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

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

Anaïs, Nadia

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 :

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

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

4 - 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

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

5 - Electrophoresis of the digestion of Bba_K11550007 and Bba_K1155003 by XBaI and PstI

Nadia

Well 1 : 6µL DNA Ladder Well 2 : 25µL of BBa_K1155003 digested by XBaI/PstII+5µl of 6X loading dye Well 3 : 25µL of BBa_K1155003 digested by XBaI/PstII+5µl of 6X loading dye Well 4 : 25µL of BBa_K1155007 digested by XBaI/PstII+5µl of 6X loading dye Well 5 : 25µL of BBa_K1155007 digested by XBaI/PstII+5µl of 6X loading dye Gel : 1%

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