Difference between revisions of "Team:Paris Saclay/Notebook/July/10"

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{{Team:Paris_Saclay/notebook_header}}
 
=Friday 10th July=
 
=Friday 10th July=
 
==Lab Work==
 
==Lab Work==
−
 
+
===PCR===
−
====PCR====
+
 
''by Coralie''
 
''by Coralie''
  
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Keep it at 4°C
 
Keep it at 4°C
  
−
====Verification of PCR products by electrophoresis====
+
===Verification of PCR products by electrophoresis===
 
''by Coralie''
 
''by Coralie''
  
 
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_10.07.15_-_PCR_tige_boucle.jpg|300px|center]]
 +
<html><i><p>Verification of PCR products, from left to right: 1. Empty, 2. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017, 6. Empty</i></p></i></html>
  
 
We confirm that the PCR was effective. We can continue the protocol.
 
We confirm that the PCR was effective. We can continue the protocol.
  
−
====Digestion====
+
===Digestion===
 
''by Coralie''
 
''by Coralie''
  
Line 43: Line 46:
 
Incubation at 37°C for 1 hour
 
Incubation at 37°C for 1 hour
  
−
====Purification of the digested PCR product====
+
===Purification of the digested PCR product===
 
''by Coralie''
 
''by Coralie''
  
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Keep the product at -20°C
 
Keep the product at -20°C
  
−
===Transformation :===
+
===Quantification of the PCR product===
 +
''by Coralie''
 +
 
 +
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET
 +
Migration 0,06A 80V
 +
 
 +
[[File:ParisSaclay_10.07.15_ARN_tige_boucle_dig_purif.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_K115017, 3. BBa_K115017, 4. Empty, 5. Empty, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty</i></p></i></html>
 +
 
 +
Concentration of the digested and purified PCR product: 50ng/µL
 +
 
 +
===Transformation===
 
''by Johan, Seong Koo''
 
''by Johan, Seong Koo''
  
−
* S03518
+
* BBa_S03518
−
* B0030
+
* BBa_B0030
−
* B0015
+
* BBa_B0015
−
* K1399005
+
* BBa_K1399005
 +
 
 +
===New culture===
 +
''by Johan''
 +
 
 +
New liquid culture of:
 +
* BBa_R0051 (2015)
 +
 
 +
5ml LB + 10μl Ampicilline + 1 bacterial colony.
 +
We incubate cultures at Room temperature for 4days.
  
 +
==Human Practices==
 +
===Meeting with Jacques Livage===
  
 
'''Members present:'''
 
'''Members present:'''
 
*Instructors : Alice.
 
*Instructors : Alice.
 
*Students : Johan, Pauline, Coralie, Audrey, Seong Koo.
 
*Students : Johan, Pauline, Coralie, Audrey, Seong Koo.
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 21:15, 18 September 2015

Friday 10th July

Lab Work

PCR

by Coralie

PCR Mix (for 3 tubes)

  • GC Buffer: 30µL
  • dNTP 10mM: 3µL
  • Forward Primer (dilution: 1/10e): 7,5µL
  • Reverse Primer (dilution: 1/10): 7,5µL
  • Template DNA K115017 (dilution 1/10e): 6µL
  • DNA polymerase Phusion: 1,5µL
  • H2O: 94,5µL

In each tube: 50µL from the mix

Cycle: Initiation: 98°C - 30seconds Cycle (30 repeats): 98°C - 10seconds / 53°C - 30seconds / 72°C - 10seconds Term.: 72°C - 5min Keep it at 4°C

Verification of PCR products by electrophoresis

by Coralie

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

ParisSaclay 10.07.15 - PCR tige boucle.jpg

Verification of PCR products, from left to right: 1. Empty, 2. DNA Ladder, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017, 6. Empty

We confirm that the PCR was effective. We can continue the protocol.

Digestion

by Coralie

We digest 2 tubes of the PCR product Mix for each tube:

  • XbaI: 1µL
  • PstI: 1µL
  • FastDigest Buffer: 2µL
  • H2O: µL
  • PCR product: 10µL

Incubation at 37°C for 1 hour

Purification of the digested PCR product

by Coralie

We use the Nucleospin kit from Magerey Nagel Keep the product at -20°C

Quantification of the PCR product

by Coralie

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

ParisSaclay 10.07.15 ARN tige boucle dig purif.jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_K115017, 3. BBa_K115017, 4. Empty, 5. Empty, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty

Concentration of the digested and purified PCR product: 50ng/µL

Transformation

by Johan, Seong Koo

  • BBa_S03518
  • BBa_B0030
  • BBa_B0015
  • BBa_K1399005

New culture

by Johan

New liquid culture of:

  • BBa_R0051 (2015)

5ml LB + 10μl Ampicilline + 1 bacterial colony. We incubate cultures at Room temperature for 4days.

Human Practices

Meeting with Jacques Livage

Members present:

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

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.