User:Dileep D. Monie/Notebook/2008-3-25: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
*Removed 5 test strains and TOP10 plates from incubator (colonies grew on all plates) | *Removed 5 test strains and TOP10 plates from incubator (colonies grew on all plates) | ||
*Prepared 250 ml Supplemented M9 Media (this is the same as the [[Endy:M9_media/supplemented|Endy Lab version]] but does not require preparation of intermediate solutions) | *Prepared 250 ml of Supplemented M9 Media (this is the same as the [[Endy:M9_media/supplemented|Endy Lab version]] but does not require preparation of intermediate solutions) | ||
**Mix the following components and autoclave at 121°C for 20 min | **Mix the following components and autoclave at 121°C for 20 min | ||
*#241 ml ddH<sub>2</sub>O | *#241 ml ddH<sub>2</sub>O | ||
Line 7: | Line 7: | ||
*#1 ml glycerol (Invitrogen 15514-011; lot no. 1248326) | *#1 ml glycerol (Invitrogen 15514-011; lot no. 1248326) | ||
*#0.5 ml 1M MgSO<sub>4</sub> (Sigma M3409; lot no. 116K6095) | *#0.5 ml 1M MgSO<sub>4</sub> (Sigma M3409; lot no. 116K6095) | ||
*#25 μl CaCl<sub>2</sub> (Teknova C0477; lot no. C047710L301Z) | *#25 μl 1M CaCl<sub>2</sub> (Teknova C0477; lot no. C047710L301Z) | ||
**Add 7.5 ml of sterile 10 mg/ml thiamine hydrochloride solution | **Add 7.5 ml of sterile 10 mg/ml thiamine hydrochloride solution | ||
*#Dissolve 100 mg thiamine hydrochloride (Sigma T1270; lot no. 037K00071) in 10 ml ddH<sub>2</sub>O | *#Dissolve 100 mg thiamine hydrochloride (Sigma T1270; lot no. 037K00071) in 10 ml ddH<sub>2</sub>O | ||
*#Filter sterilize with a 0.22 μm syringe filter | *#Filter sterilize with a 0.22 μm syringe filter | ||
**Protect Supplemented M9 Media from light | **Protect Supplemented M9 Media from light (thiamine hydrochloride is light sensitive) | ||
*Added 5ml of Supplemented M9 Media containing 20 μg/ml Kanamycin (Sigma K0254; lot no. 027K2378) to fifteen 17mm test tubes | *Added 5ml of Supplemented M9 Media containing 20 μg/ml Kanamycin (Sigma K0254; lot no. 027K2378) to fifteen 17mm test tubes | ||
*Added 5ml of Supplemented M9 Media with no antibiotic (for the TOP10 negative control cells) to three 17mm test tubes | *Added 5ml of Supplemented M9 Media with no antibiotic (for the TOP10 negative control cells) to three 17mm test tubes | ||
*Inoculated media by picking single colonies from plates (each strain was inoculated in triplicate from different colonies) | *Inoculated media by picking single colonies from plates (each strain was inoculated in triplicate from different colonies) | ||
*Grew the cultures at 37°C with shaking at 70 rpm | *Grew the cultures at 37°C with shaking at 70 rpm |
Revision as of 22:02, 25 March 2008
- Removed 5 test strains and TOP10 plates from incubator (colonies grew on all plates)
- Prepared 250 ml of Supplemented M9 Media (this is the same as the Endy Lab version but does not require preparation of intermediate solutions)
- Mix the following components and autoclave at 121°C for 20 min
- 241 ml ddH2O
- 5.64 g M9 minimal salts, 5X (Sigma M6030; lot no. 127K1574)
- 0.5 g casamino acids (Acros 61204-1000; lot no. B0123863)
- 1 ml glycerol (Invitrogen 15514-011; lot no. 1248326)
- 0.5 ml 1M MgSO4 (Sigma M3409; lot no. 116K6095)
- 25 μl 1M CaCl2 (Teknova C0477; lot no. C047710L301Z)
- Add 7.5 ml of sterile 10 mg/ml thiamine hydrochloride solution
- Dissolve 100 mg thiamine hydrochloride (Sigma T1270; lot no. 037K00071) in 10 ml ddH2O
- Filter sterilize with a 0.22 μm syringe filter
- Protect Supplemented M9 Media from light (thiamine hydrochloride is light sensitive)
- Added 5ml of Supplemented M9 Media containing 20 μg/ml Kanamycin (Sigma K0254; lot no. 027K2378) to fifteen 17mm test tubes
- Added 5ml of Supplemented M9 Media with no antibiotic (for the TOP10 negative control cells) to three 17mm test tubes
- Inoculated media by picking single colonies from plates (each strain was inoculated in triplicate from different colonies)
- Grew the cultures at 37°C with shaking at 70 rpm