Team:Paris Saclay/Notebook/August/22

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(Difference between revisions)
(→2 - Digestion of BBa_K1155000 by Spe I)
(→Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3)
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We let digestions at 37°C during 10 minutes.
We let digestions at 37°C during 10 minutes.
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===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
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===='''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 '''====
===='''1 - Gel purification of the digestion of BBa_J04450 by EcoRI/PstI '''====

Revision as of 22:33, 3 October 2013

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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

Used quantities :

  • BBa_K1155000 : 10µL
  • Buffer FD : 2µL
  • SpeI : 2µL
  • H2O : 6 µL

We let digestions at 37°C during 10 minutes.

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.

2 - Electrophoresis of gel purification of the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

Psgel12208.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_J04450 digested by EcoRI/Pst I +1µl of 6X loading dye
  • Gel : 1%

Expect sizes :

  • pSB3K3 : 2750 bp

We can't see anything with the fisrt electrophoresis that's why we made an EtOH precipitation.

3 - Ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI

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

Psgel22208.jpg
  • 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


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