"
Team:TU Darmstadt/Protocols/Protein Expression
From 2012.igem.org
(Difference between revisions)
(Created page with "<html> <link rel="stylesheet" href="https://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/css&action=raw&ctype=text/css" type="text/css" /> <div id="TUD"> <div id="...") |
|||
| Line 43: | Line 43: | ||
* [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] pET26b-Est13 |
* 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG) | * 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG) | ||
* incubator | * incubator | ||
Revision as of 17:45, 16 September 2012
Standard Protocol Protein Expression Est13
Materials
- DYT medium
- 100 mL and 3 L flasks
- E.coli [http://www.neb.com/nebecomm/products/productC3026.asp BL21 SHuffle] pET26b-Est13
- 1 mM stock of isopropyl-ß-D-thiogalactopyranosid (IPTG)
- incubator
- photometer
- kanamycin
- ice
Procedure
- 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.
