Difference between revisions of "Team:Paris Saclay/Notebook/August/25"

(Created page with "=Tuesday 25th August= ==Lab Work== ===Electrophoresis purification=== ''by Audrey'' Biobricks: * BBa_K1707004 #5 (digested by SpeI and EcoRI) * BBa_K1707022 #1 (digested b...")
 
Line 77: Line 77:
 
* 1μL plasmid (dilution 1/10 or 1/100)
 
* 1μL plasmid (dilution 1/10 or 1/100)
 
* 0,25 μL DNA Pol Phusion
 
* 0,25 μL DNA Pol Phusion
 +
 +
PCR Cycle:
 +
* 98°C - 30 seconds
 +
* 30 cycles:
 +
** 98°C - 5 seconds
 +
** 65,6°C - 30 seconds
 +
** 72°C - 3 minutes
 +
* 72°C - 10 minutes
 +
* 4°C for ever
 +
 +
====Amplification (reverse PCR) biobrick: BBa_K1707021====
 +
 +
PCR mix for each tube:
 +
* 36,75 µL H2O
 +
* 10 µL Buffer Phusion
 +
* 1 μL dNTP 10mM
 +
* 0,5 μL Forward primer
 +
* 0,5 μL Reverse primer
 +
* 1μL plasmid (dilution 1/10 or 1/100)
 +
* 0,25 μL DNA Pol Phusion
 +
 +
PCR Cycle:
 +
* 98°C - 30 seconds
 +
* 30 cycles:
 +
** 98°C - 5 seconds
 +
** 65,6°C - 30 seconds
 +
** 72°C - 3 minutes
 +
* 72°C - 10 minutes
 +
* 4°C for ever
 +
 +
====Amplification (reverse PCR) biobrick: BBa_K1707027====
 +
 +
PCR mix for each tube:
 +
* 36,75 µL H2O
 +
* 10 µL Buffer Phusion
 +
* 1 μL dNTP 10mM
 +
* 0,5 μL Forward primer
 +
* 0,5 μL Reverse primer
 +
* 1μL plasmid (dilution 1/10 or 1/100)
 +
* 0,25 μL DNA Pol Phusion
 +
 +
PCR Cycle:
 +
* 98°C - 30 seconds
 +
* 30 cycles:
 +
** 98°C - 5 seconds
 +
** 62,6°C - 30 seconds
 +
** 72°C - 2 minutes
 +
* 72°C - 10 minutes
 +
* 4°C for ever
 +
 +
 +
===Electrophoresis===
 +
''by Audrey''
 +
 +
Agarose gel 1%, migration 90V
 +
 +
We load on gel 5μL of PCR product
 +
 +
We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.
  
  

Revision as of 14:42, 12 September 2015

Tuesday 25th August

Lab Work

Electrophoresis purification

by Audrey

Biobricks:

  • BBa_K1707004 #5 (digested by SpeI and EcoRI)
  • BBa_K1707022 #1 (digested by XbaI and PstI)

Agarose gel 1%, migration 90V

We cut corresponding bands with a scalpel.

Purification

by Audrey

Biobricks:

  • BBa_K1707022 #1
  • BBa_K1707023 #1
  • BBa_K1707004 #5
  • BBa_R0040 #1

With Macherey Nagel purification kit

Quantification

by Audrey

Agarose gel 1%, migration 90V. For each sample:

  • 5 µL plasmid
  • 5 µL H2O
  • 2 µL Ladder 6x

We can conclude:

  • BBa_K1707022 #1: nothing can be seen
  • BBa_K1707023 #1: nothing can be seen
  • BBa_K1707004 #5 (SpeI + EcoRI): 20 µg/µL
  • BBa_K1707004 #5 (SpeI + PstI): 75 µg/µL
  • BBa_R0040 #1: 75 µg/µL


PCR for the Gibson experiment

by Audrey

Amplification Thermometer RNA BBa_K115017

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid BBa_K115017 v10
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 69,8°C - 30 seconds
    • 72°C - 10 seconds
  • 72°C - 10 minutes
  • 4°C for ever


Amplification (reverse PCR) biobricks with cI and cI857: BBa_K1707013, BBa_K1707019, BBa_K1707020, BBa_K1707035, BBa_K1707036

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 65,6°C - 30 seconds
    • 72°C - 3 minutes
  • 72°C - 10 minutes
  • 4°C for ever

Amplification (reverse PCR) biobrick: BBa_K1707021

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 65,6°C - 30 seconds
    • 72°C - 3 minutes
  • 72°C - 10 minutes
  • 4°C for ever

Amplification (reverse PCR) biobrick: BBa_K1707027

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 62,6°C - 30 seconds
    • 72°C - 2 minutes
  • 72°C - 10 minutes
  • 4°C for ever


Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

We load on gel 5μL of PCR product

We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.


Member present:

  • Instructors: Claire
  • Students: Audrey

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.