Team:Paris Saclay/Notebook/August/17
Contents
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:
- 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.
- Add 100 μl of Solution I. Resuspend with pipette.
- Add 200 μl of Solution II. Agitate by gently inverting the tube.
- Add 150 μl of Solution III. Agitate by inverting the tube.
- Let 10 min at 4°C.
- Centrifuge for 15 min at 11,000 rpm and retrieve the supernatant in 1.5 ml microcentrifuge tube.
- Add 100 μl of mix Phenol/Chloroform and agitate by inverting the tube.
- Centrifuge 10 min and retrieve the aqueous phase.
- Add 2 volumes of 100% ethanol.
- Let 10 min at least at 20°C.
- Centrifuge 15 min at maximum speed. Retrieve the supernatant.
- Add 1 ml of 70% ethanol.
- Centrifuge 4 min at maximum speed. Retrieve the supernatant.
- Dry
- 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
- 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
- BBa_K1707031 (08/17/2015)
Transplant
by Pauline
- 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