Difference between revisions of "Team:Paris Saclay/Notebook/July/23"

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Migration 80V
 
Migration 80V
 
Biobricks: BBa_K1707003 #4 to #9 digested by EcoRI or EcoRI+PstI
 
Biobricks: BBa_K1707003 #4 to #9 digested by EcoRI or EcoRI+PstI
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+
[[File:Paris Saclay-23.07.2015 plasmides 2.jpg|300px|center]]
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We can't conclude anything because we changed #4 in each gel and we forgot the control
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<html><p><i>Verification by digestion with EcoRI of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI and PstI, 3. #5, 4. #6, 5.#7, 6. #8, 7. #9, 8. Empty, 9. Empty, 10. Empty</i></p></html>
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[[File:Paris Saclay-23.07.2015 plasmides 1.jpg|300px|center]]
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<html><p><i>Verification by digestion with EcoRI and PstI of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI, 3. #5, 4. #6, 5.#7, 6. #8, 7. #9, 8. Empty, 9. Empty, 10. Empty</i></p></html>
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We can't conclude anything because we inverted #4 in each gel and we forgot the control
  
 
===Digestion===
 
===Digestion===
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Migration 80V
 
Migration 80V
 
Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015 digested by EcoRI or EcoRI+PstI
 
Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015 digested by EcoRI or EcoRI+PstI
−
 
+
[[File:Paris Saclay-23.07.2015 plasmides final.jpg|300px|center]]
 +
<html><p><i>Verification by digestion of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI, 3. #5 digested by EcorRI, 4. Indicator, 5.#4 digested by EcorRI and PstI, 6. #5 digested by EcorRI and PstI, 7. Indicator, 8. Empty, 9. Empty, 10. Empty</i></p></html>
 
We can confirm the BBa_K1707003 #4 and #5
 
We can confirm the BBa_K1707003 #4 and #5
  
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Agarose gel 1%, migration 70V
 
Agarose gel 1%, migration 70V
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+
[[File:ParisSaclay 23.07.2015 Purification.jpg|300px|center]]
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<html><p><i>Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707002, 3. BBa_K098997, 4. BBa_C0040</i></p></html>
 
We cut bands with a scalpel
 
We cut bands with a scalpel
  
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Agarose gel 1%
 
Agarose gel 1%
 
Migration 110V
 
Migration 110V
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+
[[File:Paris Saclay-23.07.2015 quantif.jpg|300px|center]]
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<html><p><i>Wells 2-6: Quantification, Well 7: Verification; from left to right: 1. DNA Ladder, 2. BBa_K098997, 3. BBa_B0015, 4. BBa_C0051, 5. BBa_K1707002, 6. BBa_K1707001, 7. K1707000 plasmid, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty</i></p></html>
 
We can conclude that the BBa_K1707000 PCR is ok. So the biobrick is confirmed
 
We can conclude that the BBa_K1707000 PCR is ok. So the biobrick is confirmed
  

Revision as of 09:52, 18 September 2015

Thursday 23th July

Lab Work

Soil experiment

by Audrey, Seong Koo and Johan

Plates observation and count of CFU:

  • Strain 1696:
    • Dilution 1: 490 CFU
    • Dilution 0.1: 67 CFU
    • Dilution 0.01: 0 CFU
  • Strain 1693:
    • Dilution 1: 1308 CFU
    • Dilution 0.1: 196 CFU
    • Dilution 0.01: 0 CFU
  • Strain 1320:
    • Dilution 1: 186 CFU
    • Dilution 0.1: 35 CFU
    • Dilution 0.01: 1 CFU

We take 1g of contaminated soil and treat it like yesterday to spread it on specific plates Incubation ON, 37°C

Digestion

by Coralie

Biobrick: BBa_K1707003 #4 to #9

  • Digestion by EcoRI: mix
    • 6µL EcoRI
    • 6µL Buffer FastDigest 10x
    • 36µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

  • Digestion by EcoRI + PstI: mix
    • 6µL EcoRI
    • 6µL PstI
    • 6µL Buffer FastDigest 10x
    • 30µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

Incubation 1h30, 37°C

Electrophoresis

by Johan

Agarose gel: 1% Migration 80V Biobricks: BBa_K1707003 #4 to #9 digested by EcoRI or EcoRI+PstI

Paris Saclay-23.07.2015 plasmides 2.jpg

Verification by digestion with EcoRI of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI and PstI, 3. #5, 4. #6, 5.#7, 6. #8, 7. #9, 8. Empty, 9. Empty, 10. Empty

Paris Saclay-23.07.2015 plasmides 1.jpg

Verification by digestion with EcoRI and PstI of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI, 3. #5, 4. #6, 5.#7, 6. #8, 7. #9, 8. Empty, 9. Empty, 10. Empty

We can't conclude anything because we inverted #4 in each gel and we forgot the control

Digestion

by Coralie

Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015

  • Digestion by EcoRI: mix
    • 3µL EcoRI
    • 3µL Buffer FastDigest 10x
    • 18µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

  • Digestion by EcoRI + PstI: mix
    • 3µL EcoRI
    • 3µL PstI
    • 3µL Buffer FastDigest 10x
    • 15µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

Incubation 1h30, 37°C

Electrophoresis

by Coralie

Agarose gel: 1% Migration 80V Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015 digested by EcoRI or EcoRI+PstI

Paris Saclay-23.07.2015 plasmides final.jpg

Verification by digestion of BBa_K1707003, from left to right: 1. DNA Ladder, 2. #4 digested by EcorRI, 3. #5 digested by EcorRI, 4. Indicator, 5.#4 digested by EcorRI and PstI, 6. #5 digested by EcorRI and PstI, 7. Indicator, 8. Empty, 9. Empty, 10. Empty

We can confirm the BBa_K1707003 #4 and #5


Gel purification

by Audrey

Biobricks:

  • BBa_K1707002
  • BBa_K098997
  • BBa_C0051

Agarose gel 1%, migration 70V

ParisSaclay 23.07.2015 Purification.jpg

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707002, 3. BBa_K098997, 4. BBa_C0040

We cut bands with a scalpel

Purification

by Audrey

Biobricks:

  • BBa_K098997
  • BBa_B0015
  • BBa_C0051
  • BBa_K1707002
  • BBa_K1707001

With PCR Clean up/Gel extraction kit from Macherey Nigel

Quantification and Verification

by Audrey

Biobricks:

  • Quantification:
    • BBa_K098997
    • BBa_B0015
    • BBa_C0051
    • BBa_K1707002
    • BBa_K1707001
  • Verification:
    • BBa_K1707000

Agarose gel 1% Migration 110V

Paris Saclay-23.07.2015 quantif.jpg

Wells 2-6: Quantification, Well 7: Verification; from left to right: 1. DNA Ladder, 2. BBa_K098997, 3. BBa_B0015, 4. BBa_C0051, 5. BBa_K1707002, 6. BBa_K1707001, 7. K1707000 plasmid, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty

We can conclude that the BBa_K1707000 PCR is ok. So the biobrick is confirmed

Ligation

by Coralie

  • BBa_K1707006
    • 3µL BBa_C0051
    • 2µL BBa_B0015
    • 1µL Ligase
    • 1µL Buffer 10x
    • 3µL H2O
  • BBa_K1707007
    • 8µL BBa_K098997
    • 2µL BBa_B0015
    • 1µL Ligase
    • 2µL Buffer 10x
    • 7µL H2O
  • BBa_K1707005
    • 13µL BBa_K1707002
    • 2µL BBa_K1707001
    • 1µL Ligase
    • 2µL Buffer 10x
    • 2µL H2O

Incubation ON, 4°C


Member present:

  • Instructors:Alice
  • Students: Audrey, Coralie, Pauline, Seong Koo, Johan

Back to the calendar

Contact
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France Copyright © 2015 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.