Difference between revisions of "Team:Paris Saclay/Notebook/July/16"

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{{Team:Paris_Saclay/notebook_header}}
 
=Thursday 16th July=
 
=Thursday 16th July=
−
 
+
==Lab Work==
−
 
+
===Plasmid extraction===
−
====Plasmid extraction ====
+
 
''by Johan''
 
''by Johan''
  
−
Biobrick:
+
* BBa_S03518
−
* S03518
+
* BBa_B0015
−
* B0015
+
 
Cultures from the 07/15/2015
 
Cultures from the 07/15/2015
 
With the Nucleospin Kit from Macherey Nagel
 
With the Nucleospin Kit from Macherey Nagel
  
−
====Digestion====
+
===Digestion===
 
''by Coralie''
 
''by Coralie''
 +
 +
* BBa_S03518
 +
* BBa_B0015
 +
* BBa_I13602
  
 
In each tube:
 
In each tube:
Line 19: Line 22:
 
* Buffer FastDigest (10x): 2µL
 
* Buffer FastDigest (10x): 2µL
 
* H2O: 6µL
 
* H2O: 6µL
−
 
−
Biobricks:
 
−
* S03518
 
−
* B0015
 
−
* I13602
 
  
 
Enzymes choice:
 
Enzymes choice:
−
* S03518 #1: XbaI + PstI
+
* BBa_S03518 #1: XbaI + PstI
−
* S03518 #2: SpeI + EcoRI
+
* BBa_S03518 #2: SpeI + EcoRI
−
* B0015 #1: PstI + SpeI
+
* BBa_B0015 #1: PstI + SpeI
−
* B0015 #2: XbaI + EcoRI
+
* BBa_B0015 #2: XbaI + EcoRI
−
* I13602 (x2): XbaI + Pst I
+
* BBa_I13602 (x2): XbaI + Pst I
  
 
Incubation 1h30, 37°C
 
Incubation 1h30, 37°C
  
−
====PCR====
+
===PCR===
 
''by Coralie''
 
''by Coralie''
  
−
Biobrick: R0051
+
* BBa_R0051
 
We use the rehydrated plasmid from the iGEM plate 2014
 
We use the rehydrated plasmid from the iGEM plate 2014
  
Line 57: Line 55:
 
Keep it at 4°C
 
Keep it at 4°C
  
−
====New culture====
+
===New culture===
 
''by Seong Koo''
 
''by Seong Koo''
  
 
Observation of our plates: a lot of colony in each one.  
 
Observation of our plates: a lot of colony in each one.  
 
New liquid culture of:
 
New liquid culture of:
−
* K1399005
+
* BBa_K1399005
−
* K1399019
+
* BBa_K1399019
−
* K1399023
+
* BBa_K1399023
−
* Ligation product: J23101 + K115017
+
* Ligation product: BBa_J23101 + BBa_K115017
  
 
2x 5ml LB + 10μl Chloramphenicol + 1 bacterial colony.  
 
2x 5ml LB + 10μl Chloramphenicol + 1 bacterial colony.  
 
We incubate cultures at 37°C, ON.
 
We incubate cultures at 37°C, ON.
  
−
=====Plasmid extraction =====
+
===Plasmid extraction===
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
+
* BBa_R0051
−
* R0051
+
* BBa_B0030
−
* B0030
+
 
Cultures from the 07/15/2015
 
Cultures from the 07/15/2015
 
With the Nucleospin Kit from Macherey Nagel
 
With the Nucleospin Kit from Macherey Nagel
  
−
====Digestion:====
+
===Digestion===
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
+
* BBa_R0051 (07/15/2015)
−
* R0051 (07/15/2015)
+
* BBa_R0051 (07/16/2015)
−
* R0051 (07/16/2015)
+
* BBa_B0030
−
* B0030
+
* BBa_J23101 + BBa_I13504
−
* J23101 + I13504
+
* BBa_J23106 + BBa_I13504
−
* J23106 + I13504
+
* BBa_J23117 + BBa_I13504
−
* J23117 + I13504
+
  
 
Reaction mix:
 
Reaction mix:
Line 97: Line 93:
 
* H2O: 15µL
 
* H2O: 15µL
  
−
====Electrophoresis====
+
===Electrophoresis===
 
''by Pauline''
 
''by Pauline''
  
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET
−
Migration 0,06A 80V  
+
Migration 0,06A 80V
−
 
+
[[File:ParisSaclay_16.07.15_-_digestion_vérif.jpg|300px|center]]
−
====Purification of Biobricks by electrophoresis====
+
<html><i><p>Digestion verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_R0051 (15/07), 3. BBa_R0051 (16/07), 4. BBa_B0030#1, 5. BBa_J23101+I13504#1, 6. BBa_J23106+I13504#1, 7. BBa_J23117+I13504#1, 8. Empty, 9. Empty, 10. Empty</p></i></html>
 +
===Purification of BioBricks by electrophoresis===
 
''by Coralie''
 
''by Coralie''
  
−
Biobricks:
+
* BBa_S03518
−
* S03518
+
* BBa_I13602
−
* B0015
+
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET
 
Migration 0,06A 80V  
 
Migration 0,06A 80V  
 +
[[File:ParisSaclay_16.07.2015_-_Purification.jpg|300px|center]]
 +
<html><i><p>Purificatin verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_I13602#1, 5. BBa_I13602#2, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty</p></i></html>
 
We cut interested bands with a scalpel.
 
We cut interested bands with a scalpel.
  
−
====DNA Extraction and purification====
+
===DNA Extraction and purification===
 
''by Coralie''
 
''by Coralie''
  
−
Biobricks:
+
* BBa_S03518 #1 and #2
−
* S03518 #1 and #2
+
* BBa_B0015 #1 and #2
−
* B0015 #1 and #2
+
* BBa_I13602 (x2)
−
* I13602 (x2)
+
* BBa_R0051 (from PCR)
−
* R0051 (from PCR)
+
  
 
We use the PCR Clean Up / Gel Extraction Kit from Macherey-Nagel
 
We use the PCR Clean Up / Gel Extraction Kit from Macherey-Nagel
 
We elute DNA in 30 µL.
 
We elute DNA in 30 µL.
  
−
====Quantification on Agarose Gel====
+
===Quantification on Agarose Gel===
 
''by Johan''
 
''by Johan''
  
Line 131: Line 128:
 
We load 2µL of previous purified DNA with 2 µL of DNA Loading (6x) and 8 µL of H2O
 
We load 2µL of previous purified DNA with 2 µL of DNA Loading (6x) and 8 µL of H2O
 
Migration 0,06A 80V  
 
Migration 0,06A 80V  
−
 
+
[[File:ParisSaclay_16.07.15_-_quantif_pre-ligation.jpg|300px|center]]
 +
<html><i><p>Quantification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_B0015#1, 5. BBa_B0015#2, 6. BBa_I13602#1, 7. BBa_I13602#2, 8. BBa_R0051#1, 9. BBa_BBa_R0051#2, 10. Empty, 11. Empty, 12. Empty</p></i></html>
 
We can observe that the PCR of R0051 was effective.
 
We can observe that the PCR of R0051 was effective.
 
We can quantify purified DNA:
 
We can quantify purified DNA:
−
* S03518 #1: 15 ng/µL
+
* BBa_S03518 #1: 15 ng/µL
−
* S03518 #2: 15ng/µL
+
* BBa_S03518 #2: 15ng/µL
−
* B0015 #1: 10 ng/µL
+
* BBa_B0015 #1: 10 ng/µL
−
* B0015 #2: 10 ng/µL
+
* BBa_B0015 #2: 10 ng/µL
−
* I13602 (x2): 5ng/µL
+
* BBa_I13602 (x2): 5ng/µL
−
* R0051: 10 ng/µL
+
* BBa_R0051: 10 ng/µL
−
 
+
−
 
+
  
 
'''Members present:'''
 
'''Members present:'''
 
* Instructors and advisors: Alice.
 
* Instructors and advisors: Alice.
 
* Students: Johan, Seong Koo, Coralie, Pauline
 
* Students: Johan, Seong Koo, Coralie, Pauline
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 21:16, 18 September 2015

Thursday 16th July

Lab Work

Plasmid extraction

by Johan

  • BBa_S03518
  • BBa_B0015

Cultures from the 07/15/2015 With the Nucleospin Kit from Macherey Nagel

Digestion

by Coralie

  • BBa_S03518
  • BBa_B0015
  • BBa_I13602

In each tube:

  • Plasmid: 10µL
  • Enzyme: 1µL of each enzyme
  • Buffer FastDigest (10x): 2µL
  • H2O: 6µL

Enzymes choice:

  • BBa_S03518 #1: XbaI + PstI
  • BBa_S03518 #2: SpeI + EcoRI
  • BBa_B0015 #1: PstI + SpeI
  • BBa_B0015 #2: XbaI + EcoRI
  • BBa_I13602 (x2): XbaI + Pst I

Incubation 1h30, 37°C

PCR

by Coralie

  • BBa_R0051

We use the rehydrated plasmid from the iGEM plate 2014

Reaction mix for 3 tubes:

  • GC Buffer (5x): 30µL
  • dNTP (10mM): 3µL
  • Forward Primer (1/10): 7,5µL
  • Reverse Primer (1/10): 7,5µL
  • Template DNA R0051 (2014): 5µL
  • DNA Pol Phusion: 1,5µL
  • H2O: 97,5µL

We use 50µL of that mix in each tube

Cycle: Initiation: 98°C - 30seconds Cycle (34 repeats): 98°C - 10seconds / 65°C - 30seconds / 72°C - 20seconds Term.: 72°C - 5min Keep it at 4°C

New culture

by Seong Koo

Observation of our plates: a lot of colony in each one. New liquid culture of:

  • BBa_K1399005
  • BBa_K1399019
  • BBa_K1399023
  • Ligation product: BBa_J23101 + BBa_K115017

2x 5ml LB + 10μl Chloramphenicol + 1 bacterial colony. We incubate cultures at 37°C, ON.

Plasmid extraction

by Pauline

  • BBa_R0051
  • BBa_B0030

Cultures from the 07/15/2015 With the Nucleospin Kit from Macherey Nagel

Digestion

by Pauline

  • BBa_R0051 (07/15/2015)
  • BBa_R0051 (07/16/2015)
  • BBa_B0030
  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

Reaction mix:

  • Plasmid: 2µL
  • EcoRI: 0,5µL
  • PstI: 0,5µL
  • Buffer FastDigest (10x): 2µL
  • H2O: 15µL

Electrophoresis

by Pauline

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

ParisSaclay 16.07.15 - digestion vérif.jpg

Digestion verification, from left to right: 1. DNA Ladder, 2. BBa_R0051 (15/07), 3. BBa_R0051 (16/07), 4. BBa_B0030#1, 5. BBa_J23101+I13504#1, 6. BBa_J23106+I13504#1, 7. BBa_J23117+I13504#1, 8. Empty, 9. Empty, 10. Empty

Purification of BioBricks by electrophoresis

by Coralie

  • BBa_S03518
  • BBa_I13602

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

ParisSaclay 16.07.2015 - Purification.jpg

Purificatin verification, from left to right: 1. DNA Ladder, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_I13602#1, 5. BBa_I13602#2, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty

We cut interested bands with a scalpel.

DNA Extraction and purification

by Coralie

  • BBa_S03518 #1 and #2
  • BBa_B0015 #1 and #2
  • BBa_I13602 (x2)
  • BBa_R0051 (from PCR)

We use the PCR Clean Up / Gel Extraction Kit from Macherey-Nagel We elute DNA in 30 µL.

Quantification on Agarose Gel

by Johan

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET We load 2µL of previous purified DNA with 2 µL of DNA Loading (6x) and 8 µL of H2O Migration 0,06A 80V

ParisSaclay 16.07.15 - quantif pre-ligation.jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_B0015#1, 5. BBa_B0015#2, 6. BBa_I13602#1, 7. BBa_I13602#2, 8. BBa_R0051#1, 9. BBa_BBa_R0051#2, 10. Empty, 11. Empty, 12. Empty

We can observe that the PCR of R0051 was effective. We can quantify purified DNA:

  • BBa_S03518 #1: 15 ng/µL
  • BBa_S03518 #2: 15ng/µL
  • BBa_B0015 #1: 10 ng/µL
  • BBa_B0015 #2: 10 ng/µL
  • BBa_I13602 (x2): 5ng/µL
  • BBa_R0051: 10 ng/µL

Members present:

  • Instructors and advisors: Alice.
  • Students: Johan, Seong Koo, Coralie, Pauline

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.