Team:Paris Saclay/Notebook/July/9

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='''Notebook : July 9'''=
 
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==='''Summary :'''===
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='''Notebook : July 9'''=
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For regulation system :
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*extraction of BioBrick BBa_K1155000 in concentrated medium for further DNA sequencing.
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*the terminator BBa_B0010 in PSB1A2 plasmidwas extracted form iGEM plate kit 2013 and  was transformed into competent cells and then was cultured on solid medium
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*the plasmid PSB3K3(BBa_J04450) was extracted form iGEM plate kit 2013 and transformed into competent cells and then was cultured on solid medium for confirmation
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For sensor system:
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*A series of digestion, ligation were performed for BioBrick BphR2, BphR1, BphA1
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=='''Lab work'''==
=='''Lab work'''==
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<u>DNA purification</u>
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===='''Objective : obtaining biobricks in pSB3K3'''====
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<p>See protocol DNA purification
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Briobrick promoter BphR1, BphR2, BphA1 were purified.</p>
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<u>Restriction digestion</u>
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<p>We had 5 samples for digestion: 3 extracts, plasmid PSB1C3 and Plasmid PSB3K3(extracted from plate kit 2013 6F ).</p>
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<p>For the plasmid (PSB1C3 and PSB3K3):</p>
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*Plasmid : 4µl
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*(orange): 0.8µl
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*EcoR I: 0.5µl
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*PST I:0.5µl
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*H2O:2.2µl
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*Total:8µl
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<p>For the DNA extract:</p>
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===='''1 - Transformation of BBa_J04450 in DH5α'''====
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*DNA: 15µl
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*Buffer(orange): 3µl
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*EcoR I: 0.75µl
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*PST I:0.75µl
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*H2O:10.5µl
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*Total:30µl
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<p>After the digestion, superfluous enzyme was removed by Ethanol precipitation method.
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Then we suspended them with 10µl H2O.</p>
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<u>Quantification</u><br>
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Anaïs
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{| border="1" align="center"
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|-
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|DNA
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|concentration(ng/µl)
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|260/280
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|-
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|BphR2
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|24.2
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|1.97
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|-
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|BphA1
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|108.3
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|1.85
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|-
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|BphR1
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|97.9
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|1.82
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|-
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|PSB1C3
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|36.1
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|1.77
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|-
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|PSB3K3
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|19.4
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|1.77
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|}
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<u>Ligation</u>
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Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
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<p>Common way for ligation.</p>
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*Plasmid : 2µl
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*Bph : 2µl
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*Buffer : 2µl
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*T4 ligase : 12µl
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*H2O : about 7µl
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*Total : 20µl
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<br>
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<u>Transformation</u>
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<p>See protocol transformation
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terminator BBa_B0010 in PSB1A2 plasmid and plasmid PSB3K3was transformed into competent cells and then was cultured on solid medium.</p>
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==='''B - PCB sensor system'''===
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===='''Objective : obtaining BBa_K1155001, BBa_K1155002 and BphR2 protein'''====
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===='''1 - PCR purification of BphR1, BphR2 and BphA1'''====
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Zhou
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We did a PCR amplification for BphR1, BphR2 and BphA1 genes from strain ''Pseudomonas pseudoalcaligenes''. Products were good and purified following the protocol.
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Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
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===='''2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3'''====
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Abdou, Anaïs
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Used quantities :
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* BphA1 :
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** DNA : 8µL
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** Buffer FD : 1.6µL
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** EcoRI FD : 1µL
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** PstI FD : 1µL
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** H2O : 4.4µL
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* BphR1, BphR2 :
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** DNA : 15µL
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** Buffer FD : 3µL
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** EcoRI FD : 0.75µL
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** PstI FD : 0.75µL
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** H2O : 10.5µL
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* pSB1C3 :
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** pSB1C3 : 4µL
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** Buffer orange : 0.8µL
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** EcoRI : 0.5µL
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** PstI : 0.5µL
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** H2O : 2.2µL
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We let our digestion 1h30 at 37°C.
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===='''3 - Inactivation of EcoRI/PstI used for the digestion of PCR products : BphR1, BphR2, BphA1 and pSB1C3'''====
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Anaïs, Sheng
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Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
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Nanodrop :
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* BphA1 : 108.3ng/µL
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* BphR1 : 97.9ng/µL
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* BphR2 : 24.2ng/µL
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* pSB1C3 : 36.1ng/µL
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===='''4 - Ligation of BphA1, BphR1, BphR2 and pSB1C3'''====
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Zhou
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Used quantities :
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* pSB1C3 : 2µL
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* DNA : 2µL
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* Buffer ligase : 2µL
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* Ligase : 1µL
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* H2O : 13µL
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=='''Human Practices'''==
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We made the thrid meeting about open source : [[Team:Paris_Saclay/opensourcereflexion|Open Source Reflexion]]
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|[[Team:Paris Saclay/Notebook/July/10|<big>Next day</big>]]
|[[Team:Paris Saclay/Notebook/July/10|<big>Next day</big>]]
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Latest revision as of 23:56, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 9

Lab work

Objective : obtaining biobricks in pSB3K3

1 - Transformation of BBa_J04450 in DH5α

Anaïs

Protocol : Bacterial transformation


B - PCB sensor system

Objective : obtaining BBa_K1155001, BBa_K1155002 and BphR2 protein

1 - PCR purification of BphR1, BphR2 and BphA1

Zhou

We did a PCR amplification for BphR1, BphR2 and BphA1 genes from strain Pseudomonas pseudoalcaligenes. Products were good and purified following the protocol.

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

2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3

Abdou, Anaïs

Used quantities :

  • BphA1 :
    • DNA : 8µL
    • Buffer FD : 1.6µL
    • EcoRI FD : 1µL
    • PstI FD : 1µL
    • H2O : 4.4µL
  • BphR1, BphR2 :
    • DNA : 15µL
    • Buffer FD : 3µL
    • EcoRI FD : 0.75µL
    • PstI FD : 0.75µL
    • H2O : 10.5µL
  • pSB1C3 :
    • pSB1C3 : 4µL
    • Buffer orange : 0.8µL
    • EcoRI : 0.5µL
    • PstI : 0.5µL
    • H2O : 2.2µL

We let our digestion 1h30 at 37°C.

3 - Inactivation of EcoRI/PstI used for the digestion of PCR products : BphR1, BphR2, BphA1 and pSB1C3

Anaïs, Sheng

Protocol : Ethanol precipitation

Nanodrop :

  • BphA1 : 108.3ng/µL
  • BphR1 : 97.9ng/µL
  • BphR2 : 24.2ng/µL
  • pSB1C3 : 36.1ng/µL

4 - Ligation of BphA1, BphR1, BphR2 and pSB1C3

Zhou

Used quantities :

  • pSB1C3 : 2µL
  • DNA : 2µL
  • Buffer ligase : 2µL
  • Ligase : 1µL
  • H2O : 13µL


Human Practices

We made the thrid meeting about open source : Open Source Reflexion


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