Minimal Media cell growth- ¹³C and/or ¹⁵N Labeled Protein Preparation

1. The day before you start a large ¹⁵N-Labeled protein Preparation, start 5mL LB/AMP cultures in the morning (~8-9AM) for each 4L flask that will be used. In the meantime make sure the next step has been completed.

2. Prepare the following buffers and flasks if they are not already made. 0.2M ZnCl₂, 1M MgSO₄, 0.2M CaCl₂, 10X M9 salts, 4L flasks with 900mL ddH₂O, and 1L flasks with 90mL ddH₂O should all be autoclaved separately.

0.2M ZnCl2	1M MgSO4	0.2M CaCl2
∑ 100mL ddH2O	∑ 100mL ddH2O	∑ 100mL ddH2O
∑ 2.72 g ZnCl2	∑ 24.65 g MgSO4	∑ 2.94 g (CaCl2f2H2O) or 2.22 g (CaCl2)
∑ autoclave/cool	∑ autoclave/cool	∑ autoclave/cool
10X M9 Salts (1L)	1L Minimal Media (use 4L flask)	100 mL Minimal Media (use 1L flask)
∑ 67.9 g Na2PO4	∑ 900mL ddH2O	∑ 90mL ddH2O
∑ 30.0 g KH2PO4	∑ autoclave/cool	∑ autoclave/cool
∑ 5.0 g NaCl
∑ pH 7.2
∑ autoclave/cool

3. Around 4-5 PM remove the 5mL LB cultures from the shaker and spin them down for 10 min and pour off LB. While they are being spun down add the remaining chemicals to the 100mL Minimal Media starter cultures. There should be one 100mL Minimal Media starter culture per 1L Minimal Media flask being used. Grow the 100mL starter culture overnight at 37⁰C.

100 mL Minimal Media
Already done	∑ 90mL ddH2O
Already done	∑ autoclave/cool
Add	∑ 10mL 10X M9 salts
Add	∑ 100uL MgSO4
Add	∑ 100uL 1000X Trace metals
Add	∑ 50uL 0.2M ZnCl2
Add	∑ 50uL 0.2M CaCl2
Add	∑ 0.1 g NH4Cl2
Add	∑ 0.4 g Glucose
Check (one flask)	∑ pH ~7.2
Add	∑ 1mL Vitamin mix
Add	∑ 150 uL 50mg/mL Amp
Resuspend	∑ One 5mL starter culture

4. The following morning, combine all the 100mL Minimal Media starter cultures and spin down cells at 6,000g for 30 min. In the meantime prepare the 1L Minimal Media flasks for cell growth:

1L Minimal Media
Already done	∑ 900mL ddH2O
Already done	∑ autoclave/cool
Add	∑ 100mL 10X M9 salts
Add	∑ 1mL MgSO4
Add	∑ 1mL 1000X Trace metals
Add	∑ 500uL 0.2M ZnCl2
Add	∑ 500uL 0.2M CaCl2
Add	∑ 1 g NH4Cl2 or ^15NH4Cl2
Add	∑ 4 g Glucose or ^13C-Glucose
Check (one flask)	∑ pH ~7.2
Add	∑ 10mL Vitamin mix
Add	∑ 1.5mL 50mg/mL Amp

Once the 1L Minimal Media is prepared take 10mL from each flask to resuspend the cell pellet. Aliquot 10mL of resuspended cell pellet media into each 4L flask.

5. Take an initial OD reading at 600nm.

6. Grow cells at 37⁰C until you reach an OD of 0.6. Make sure to check the OD readings approximately every hour until the OD=0.6.

7. Once the cells have reached 0.6, induce protein expression with 1mL of 1M IPTG per 1L of Minimal Media.

8. Grow for 3-4 hours post-induction. Based on 200mL protein expression assays you should know the time at which your protein reaches max expression and max OD.

9. Collect cells at 6,000g for 30 minutes.