Difference between revisions of "Team:Paris Saclay/Notebook/August/17"
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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. | 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. | ||
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2. Add 100 μl of Solution I. Resuspend with pipette. | 2. Add 100 μl of Solution I. Resuspend with pipette. | ||
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3. Add 200 μl of Solution II. Agitate by gently inverting the tube. | 3. Add 200 μl of Solution II. Agitate by gently inverting the tube. | ||
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4. Add 150 μl of Solution III. Agitate by inverting the tube. | 4. Add 150 μl of Solution III. Agitate by inverting the tube. | ||
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5. Let 10 min at 4°C. | 5. Let 10 min at 4°C. | ||
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6. Centrifuge for 15 min at 11,000 rpm and retrieve the supernatant in 1.5 ml microcentrifuge tube. | 6. Centrifuge for 15 min at 11,000 rpm and retrieve the supernatant in 1.5 ml microcentrifuge tube. | ||
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7. Add 100 μl of mix Phenol/Chloroform and agitate by inverting the tube. | 7. Add 100 μl of mix Phenol/Chloroform and agitate by inverting the tube. | ||
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8. Centrifuge 10 min and retrieve the aqueous phase. | 8. Centrifuge 10 min and retrieve the aqueous phase. | ||
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9. Add 2 volumes of 100% ethanol. | 9. Add 2 volumes of 100% ethanol. | ||
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10. Let 10 min at least at 20°C. | 10. Let 10 min at least at 20°C. | ||
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11. Centrifuge 15 min at maximum speed. Retrieve the supernatant. | 11. Centrifuge 15 min at maximum speed. Retrieve the supernatant. | ||
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12. Add 1 ml of 70% ethanol. | 12. Add 1 ml of 70% ethanol. | ||
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13. Centrifuge 4 min at maximum speed. Retrieve the supernatant. | 13. Centrifuge 4 min at maximum speed. Retrieve the supernatant. | ||
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14. Dry | 14. Dry | ||
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15. Resuspend in 50 μl of TE/RNAse for a plasmid. | 15. Resuspend in 50 μl of TE/RNAse for a plasmid. | ||
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* 5 μl of RNase (Ci = 10 mg/ml) | * 5 μl of RNase (Ci = 10 mg/ml) | ||
* 1 ml of TE from Macherey-Nagel kit | * 1 ml of TE from Macherey-Nagel kit | ||
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===New culture=== | ===New culture=== | ||
Revision as of 18:01, 10 September 2015
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:
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
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France
Copyright © 2015 iGEM Paris Saclay. *This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.