Team:Paris Saclay/Notebook/July/10
Contents
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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