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

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

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.

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