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Team:TU Darmstadt/Protocols/Protein Expression
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| - | == Standard Protocol Protein Expression | + | == Standard Protocol Protein Expression== |
=== Materials === | === Materials === | ||
* [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] | * [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] | ||
* 100 mL and 3 L flasks | * 100 mL and 3 L flasks | ||
| - | * ''E.coli'' [http://www.neb.com/nebecomm/products/productC3026.asp BL21 SHuffle] pET26b-Est13 | + | * ''E.coli'' [http://www.neb.com/nebecomm/products/productC3026.asp BL21 SHuffle] for pET26b-Est13 and [http://www.neb.com/nebecomm/products/productC3026.asp BmH7118] for pEX-FsC |
* 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG) | * 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG) | ||
* incubator | * incubator | ||
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* kanamycin | * kanamycin | ||
* ice | * ice | ||
| - | === | + | === Protein Expression Est13 === |
# Inoculation of 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] in the 100 mL flask with ''E.coli'' BL21 SHuffle containing pET26b plasmid with the sequence for the protein Est13. Addition of the 15 µL kanamycin to the medium. | # Inoculation of 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] in the 100 mL flask with ''E.coli'' BL21 SHuffle containing pET26b plasmid with the sequence for the protein Est13. Addition of the 15 µL kanamycin to the medium. | ||
# Incubation at 180 repulsion per minute (rpm) at 30°C to an OD<sub>600</sub>= 4. | # Incubation at 180 repulsion per minute (rpm) at 30°C to an OD<sub>600</sub>= 4. | ||
# Transferation of the starter culture into 1 L [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] in a 3 L flask resulting in an OD<sub>600</sub>= 0.2. Addition of 300 µL kanamycin. | # Transferation of the starter culture into 1 L [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] in a 3 L flask resulting in an OD<sub>600</sub>= 0.2. Addition of 300 µL kanamycin. | ||
| + | # Incubation to an OD<sub>600</sub>= 0.6 at 180 rpm and 30°C. | ||
| + | # Incubation for 15 min. on ice. | ||
| + | # Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM). | ||
| + | # Incubation of the cell suspension over night at 180 rpm at 16°C. | ||
| + | |||
| + | === Protein Expression FsC === | ||
| + | # Inoculation of 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT medium] in the 100 mL flask with ''E.coli'' [http://www.neb.com/nebecomm/products/productC3026.asp BmH7118] containing pEX plasmid with the sequence for the protein FsC. Addition of the 15 µL kanamycin to the medium. | ||
| + | # Incubation at 180 repulsion per minute (rpm) at 30°C to an OD<sub>600</sub>= 4. | ||
| + | # Transferation of the starter culture into 1 L [[DYT medium]] in the 3 L flask with that results in an OD<sub>600</sub>= 0.2. Addition of 300 µL kanamycin. | ||
# Incubation to an OD<sub>600</sub>= 0.6 at 180 rpm and 30°C. | # Incubation to an OD<sub>600</sub>= 0.6 at 180 rpm and 30°C. | ||
# Incubation for 15 min. on ice. | # Incubation for 15 min. on ice. | ||
# Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM). | # Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM). | ||
# Incubation of the cell suspension over night at 180 rpm at 16°C. | # Incubation of the cell suspension over night at 180 rpm at 16°C. | ||
Revision as of 17:54, 16 September 2012
Contents |
Standard Protocol Protein Expression
Materials
- DYT medium
- 100 mL and 3 L flasks
- E.coli [http://www.neb.com/nebecomm/products/productC3026.asp BL21 SHuffle] for pET26b-Est13 and [http://www.neb.com/nebecomm/products/productC3026.asp BmH7118] for pEX-FsC
- 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG)
- incubator
- photometer
- kanamycin
- ice
Protein Expression Est13
- Inoculation of 50 mL DYT medium in the 100 mL flask with E.coli BL21 SHuffle containing pET26b plasmid with the sequence for the protein Est13. Addition of the 15 µL kanamycin to the medium.
- Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4.
- Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2. Addition of 300 µL kanamycin.
- Incubation to an OD600= 0.6 at 180 rpm and 30°C.
- Incubation for 15 min. on ice.
- Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).
- Incubation of the cell suspension over night at 180 rpm at 16°C.
Protein Expression FsC
- Inoculation of 50 mL DYT medium in the 100 mL flask with E.coli [http://www.neb.com/nebecomm/products/productC3026.asp BmH7118] containing pEX plasmid with the sequence for the protein FsC. Addition of the 15 µL kanamycin to the medium.
- Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4.
- Transferation of the starter culture into 1 L DYT medium in the 3 L flask with that results in an OD600= 0.2. Addition of 300 µL kanamycin.
- Incubation to an OD600= 0.6 at 180 rpm and 30°C.
- Incubation for 15 min. on ice.
- Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).
- Incubation of the cell suspension over night at 180 rpm at 16°C.
