Team:Paris Saclay/Notebook/August/17

Monday 17th August

Lab Work

Ligation

by Pauline

• BBa_K1707031: BBa_K1707004 and BBa_R0040
  • 12,3 µL BBa_K1707004
  • 2,5 µL BBa_R0040
  • 2 µL Ligase
  • 2 µL Buffer
  • 1,2 µL H2O

Incubation 3h, 4°C

Plasmid extraction

by Pauline

BIobricks:

• BBa_K1707035 #1 and #2
• BBa_K1707036 #1 and #2

With Sylvie Lautru's Protocol:

1. Take 2 ml of bacteria culture in a 2 ml microcentrifuge tube (eppendorf), prepare one sample. Centrifuge the culture for 30s at 11,000 rpm. Discard supernatant and remove as much of the liquid as possible. Repeat this with adding 2 ml of bacteria culture in the same tube.

2. Add 100 μl of Solution I. Resuspend with pipette.

3. Add 200 μl of Solution II. Agitate by gently inverting the tube.

4. Add 150 μl of Solution III. Agitate by inverting the tube.

5. Let 10 min at 4°C.

6. Centrifuge for 15 min at 11,000 rpm and retrieve the supernatant in 1.5 ml microcentrifuge tube.

7. Add 100 μl of mix Phenol/Chloroform and agitate by inverting the tube.

8. Centrifuge 10 min and retrieve the aqueous phase.

9. Add 2 volumes of 100% ethanol.

10. Let 10 min at least at 20°C.

11. Centrifuge 15 min at maximum speed. Retrieve the supernatant.

12. Add 1 ml of 70% ethanol.

13. Centrifuge 4 min at maximum speed. Retrieve the supernatant.

14. Dry

15. Resuspend in 50 μl of TE/RNAse for a plasmid.

Solution I:

• Tris final 25 mM
• EDTA final 10 mM
• Glucose final 50 mM

Solution II:

• SDS final 1%
• NaOH final 0.1M

Solution III: CH3COOK 3M (pH 4.8):

• 28.5 ml H2O
• 60 ml CH3COOK 5M
• 11.5 ml pure CH3COOCH

TE/RNAse:

• 5 μl of RNase (Ci = 10 mg/ml)
• 1 ml of TE from Macherey-Nagel kit

New culture

by Pauline

Biobricks:

• BBa_K1707022 #1 and #2
• BBa_K1707023 #1 and #2
• BBa_K1707034 #1 and #2

We put 2 new clones in 5mL LB + 5µL Chloramphenicol

Transformation

by Pauline

Biobricks:

• BBa_K1707031 (08/17/2015)

Transplant

by Pauline

Biobrick: BBa_K1707031 (08/11/2015) We transplant 9 clones on a LB plate + Cm divided on 9.

Inoculation

by Pauline

3 strains: 1320; 1693; 1696

in 5mL LB with appropriate antibiotic

Member present:

• Instructors: Claire
• Students: Pauline

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