Team:Paris Saclay/Notebook/July/16

Thursday 16th July

Plasmid extraction

by Johan

Biobrick:

  • S03518
  • B0015

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

Digestion

by Coralie

In each tube:

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

Biobricks:

  • S03518
  • B0015
  • I13602

Enzymes choice:

  • S03518 #1: XbaI + PstI
  • S03518 #2: SpeI + EcoRI
  • B0015 #1: PstI + SpeI
  • B0015 #2: XbaI + EcoRI
  • I13602 (x2): XbaI + Pst I

Incubation 1h30, 37°C

PCR

by Coralie

Biobrick: 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:

  • K1399005
  • K1399019
  • K1399023
  • Ligation product: J23101 + K115017

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

Plasmid extraction

by Pauline

Biobricks:

  • R0051
  • B0030

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

Digestion:

by Pauline

Biobricks:

  • R0051 (07/15/2015)
  • R0051 (07/16/2015)
  • B0030
  • J23101 + I13504
  • J23106 + I13504
  • J23117 + 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

Purification of Biobricks by electrophoresis

by Coralie

Biobricks:

  • S03518
  • B0015

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V We cut interested bands with a scalpel.

DNA Extraction and purification

by Coralie

Biobricks:

  • S03518 #1 and #2
  • B0015 #1 and #2
  • I13602 (x2)
  • 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

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

  • S03518 #1: 15 ng/µL
  • S03518 #2: 15ng/µL
  • B0015 #1: 10 ng/µL
  • B0015 #2: 10 ng/µL
  • I13602 (x2): 5ng/µL
  • R0051: 10 ng/µL


Members present:

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