Team:Paris Saclay/Notebook/August/22

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Notebook : August 22

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Plasmid extraction of Bba_K1155000 from DH5α

Nguyen

Protocol : High copy plamid extraction

Nanodrop :

  • Bba_K1155000 : 175ng/µL

The extraction was good. We will digested the plasmid.

2 - Digestion of Bba_K1155000 by SpeI

Nguyen

Used quantities :

  • Bba_K1155000 : 10µL
  • Buffer FD : 2µL
  • Spe I : 2µL
  • H2O : 6 µL

We let digestions at 37°C during 10 minutes ??????

QU'EN A T'ON FAIT PAR LA SUITE ?????????

((((((((((((((((((====Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3====

1 - Gel purification of the digestion of Bba_J04450 by EcoRI/PstI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • PSB3K3 : 4ng/µL

The Nanodrop gives us a very few quantity of PSB3K3 so we decided to check it with a first electrophoresis.

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of Bba_J04450 digested by EcoRI/PstI+1µl of 6X loading dye
  • Gel : 1%

Expect sizes :

  • PSB3K3 : 2750 bp

We can see anything with the fisrt electrophoresis that's why we made an EtOH precipitation.

Protocol : EtOH precipitation

We used 34µL of DNA. ))))))))))))))))))))))

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

Objective : obtaining BphR2 protein

1 - 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 :

Pstest.jpg

2 - Electrophoresis of PCR product : BphR2 Part I

Damir

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 40µL of BphR2 Part I+8µl of 6X loading dye
  • Gel : 0.8%

Expected sizes : Bphr2 Part I : 178 kb

We obtain fragments at the right size. We can purify it.

3 - Gel purification of PCR product : BphR2 Part I

Damir

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • RBS-BphR2 Part I, tube 1 : 42ng/µL
  • RBS-BphR2 Part I, tube 2 : 75ng/µL

CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!


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