Team:Aix-Marseille/Manipulation
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CALENDRIER DESACTIVE EN ATTENDANT DE FINIR lA RETRANSCRIPTION
Contents
Day 1 : 06/06/2017
Cleaning the laboratory, then recovery and installation of the equipment. Starting culture of strains TG1 and DH5α. We launched precultures from cryotubes ( sampling with 1 rod and then deposition in an erlenmeyer containing LB media ). Incubation at 37 ° C.
Day 2 : 07/06/2017
Strains cultivation:
Recovery of strains cultivated the day before: DH5α and TG1 To check the concentrations, the OD is measured. For doing so, the culture must be diluted because the optimum measurement of the apparatus is between 0.1 and 0.8. The cultures in the tank are diluted to 1/10 with distilled water. $$\text{ Our results } : \text{ TG1: 5.16 } / \text{ DH5α: 5.21 }$$ To calculate the volume to be taken for our 100 mL we do: $$\frac{\text{OD wanted}}{\text{OD obtained before dilution}} \times \text{Volume} = \frac{0.1}{5.16} \times 100 \simeq 2 \text{ mL}$$ E. coli being aerobic, the solution is stored in LB in a Erlenmeyer flask of 5 × volume, so 500 mL and incubate for 1 h.
Glycerolization of strains:
Putting the strains in glycerol protect them from the cold, necessary when one will freeze at -80 ° C so that the cells do not collapse. 40% Glycerol in 1.8mL cryotubes → 0.9 of glycerol and 0.9 of strains. We will freeze the cells in exponential phases in order to make our cells competent, ie able to incorporate DNA.
Competent bacteria:
To make competent bacteria, it is necessary to take them in exponential growth phase. A quantity of bacterium is thus taken which is placed in LB medium and then incubated for 1 hour at 37 degree Celsius. An OD value of 0.1 is desired in 100 mL. For TG1-> 1.94 mL For DH5α-> 1.92 mL
Mesuring OD after 1h40:
TG1 - 0.7
DH5α - 0.5
The cultures were thus recovered and were divided into falcons (50 ml each) and placed in ice. (For each of the two strains).
Preparation of the buffers: Solution TBF1 and TBF2
Preparation of buffer solutions:
KAc: 50 mL → 4.9 g
MnCl2: 200 mL → 19.79 g
KCl: 200 mL → 14.91 g
MOPS: 50 mL → 2.09 g
Each solution was autoclaved.
Then we Made two solutions necessary for making the competent cells.
$$ Buffer 1 - \text{ 80 mL: }
\text{ 2.4 } \text{ mL } \text{KAc} \text{ [1 M] }, \text{8 mL} \text{ MnCl2 } \text{ [0.5 M] } , \text{8 mL} \text{ KCl } \text{ [1 M] } , \text{ 8 mL } \text{ CaCl2 } \text{ [0.1 M] }, \text{ 15 mL } \text{ Gly } \text{ [80%] } , \text{ → } \text{ 38.6 mL } \text{ H2O } $$
$$ Buffer 2 - \text{8 mL:}
\text{ 400 } \text{ μL } \text{ MOPS } \text{ [0.2M] }, \text{ 6 mL } \text{ CaCl2 } \text{ [0.1 M] }, \text{ 1.5 } \text{ mL }
\text{ Gly } \text{ [80%] }, \text{ 80 μL } \text{ KCl } \text{ [1 M] }, \text{ 500 μL } \text{ H2O}$$
To make competent cells, we ALWAYS work at low temperature (4 ° C) and in sterile medium. The buffers thus prepared were stored in ice.
Centrifugation of the cell culture 10 min at 3500 rpm at 4 ° C. The pellet must then be resuspended (gently) in 80 ml of Tbf1 (20 ml as 50 ml of culture in each falcon).
3500 rpm centrifugation for 5 minutes.
Then resuspension of the pellet in 2 mL of Tbf2.
Let incubate for 15 minutes in ice and then aliquot 200 μL of bacteria solution in a cryotube and store at -80 ° C. These aliquots are at I8 for competent DH5 alpha and I9 for competent TG1.
The remainder is stored at -20 ° C in labeled falcons.
After the first centrifugation was carried out in a cold (non-sterile) chamber, we re-started precultures from those in the morning: 100 μL of culture were placed in 10 ml of LB medium and then incubated at 37 ° C. overnight.
The buffer Tbf1 and Tbf2 have also been prepared to be ready so we can restart the manipets in case of failure.
Day 3: 08/06/17
Preparation for transformation : In four steps
For the petri dish LB agar, take a bottle of LB agar ( 400mL for 20 boxes ) and put it in the microwave to liquefy. Heat with microwave 300 watt ( no more ) for 19 minutes with unscrewed plug.
Organizational axis :
Subjects organisation
| Killer red | Crispr Cas9 | P3 | Génome M13 | Measurement | DEPS | QS |
| Robin | Flora/Soraya | Camille | Camille/Thibault | Lisa | Hussein | Jeremy
Flora Ilann
|
Small theoretical session of Sandra on the manipulations:
=== 1.Transformation: === Transformation is a technic used to make the DNA "enter" into the competent cell by destabilizing the membrane:
Step 1: Contact: The cells and plasmids (which are recovered in the iGEM kit)
Step 2: Heat shock: More permeable membrane that will make the DNA enter: 45 seconds at 42 ° C
Step 3: Expression: Cells are allowed to express the inserted genes (including antibiotic resistance which serve as a test)
There are two types of antibiotics: bacteriostatic (ampicillin, which freezes growth), bactericidal (which kills)
Step 4: Spread on antibiotic (petri dish), that will eliminate the bacteria which have not assimilated the gene
2.Verification of contamination
Negative control: 3 antibiotics, if the bacteria resist: contaminated
3.Cloning
We want to integrate an insert, for this, we need the restriction enzymes
4. Biobrick - iGEM
S and X are compatible
