From 2007.igem.org

Revision as of 17:27, 19 July 2007 (view source) MaijaP (Talk | contribs) (→Transforming BioBricks) ← Older edit		Revision as of 17:30, 19 July 2007 (view source) MaijaP (Talk | contribs) Newer edit →
Line 10:		Line 10:
	#Spread the reaction on kan/carb LB plates.		#Spread the reaction on kan/carb LB plates.
	#Grow +37 ºC overnight.		#Grow +37 ºC overnight.
		+
		+	== Minimal Media and Trace Elements ==
		+	=== Trace Elements ===
		+	#In 800ml dH2O dissolve 5g Na2 EDTA and convert to pH7.
		+	#Add the following in order, correcting to pH7 after each.
		+	#*FeCl3 (.H2O) 0.5 (0.83g)
		+	#*ZnCl2 0.05g
		+	#*CuCl2 0.01g
		+	#*CoCl2.6H20  0.01g
		+	#*H3BO3 0.01g
		+	#*MnCl2.6H2O (.4H2O) 1.6g (1.35g)
		+	#Make up to 1 litre, autoclave and store at 4 °C.
		+
		+	=== M9 ===
		+	#Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds
		+	#*6g Na2HPO4
		+	#*3g KH2PO4
		+	#*0.5g NaCl
		+	#The add
		+	#*1ml 1M MgSO4
		+	#*200ul 1M CaCl2
		+	#*1 ml Thiamine (40mg ml-1 stock)
		+	#*10 ml Trace Elements
		+	#Also add as necessary
		+	#*15ml (13C-) Glucose (20% stock) (gives 0.3% final)
		+	#*1g (15) NH4Cl (if using M9-)
		+
		+	=== Tips ===
		+	#Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction.

---

Revision as of 17:30, 19 July 2007

Contents 1 Transforming BioBricks 2 Minimal Media and Trace Elements 2.1 Trace Elements 2.2 M9 2.3 Tips

Transforming BioBricks

1. Split the competent E. coli cells (~75ul) in two.
2. Competent cells (~33ul) into eppendorfs on ice.
3. Add 1µl of plasmid DNA.
4. Incubate on ice for at least 5 mins.
5. Heat shock +42 ºC for 45 secs.
6. Back on ice for 2 mins to reduce damage.
7. Add 1ml of soc media.
8. Incubate +37ºC in a shaker for 1 hour.
9. Spread the reaction on kan/carb LB plates.
10. Grow +37 ºC overnight.

Minimal Media and Trace Elements

Trace Elements

1. In 800ml dH2O dissolve 5g Na2 EDTA and convert to pH7.
2. Add the following in order, correcting to pH7 after each.
   • FeCl3 (.H2O) 0.5 (0.83g)
   • ZnCl2 0.05g
   • CuCl2 0.01g
   • CoCl2.6H20 0.01g
   • H3BO3 0.01g
   • MnCl2.6H2O (.4H2O) 1.6g (1.35g)
3. Make up to 1 litre, autoclave and store at 4 °C.

M9

1. Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds
   • 6g Na2HPO4
   • 3g KH2PO4
   • 0.5g NaCl
2. The add
   • 1ml 1M MgSO4
   • 200ul 1M CaCl2
   • 1 ml Thiamine (40mg ml-1 stock)
   • 10 ml Trace Elements
3. Also add as necessary
   • 15ml (13C-) Glucose (20% stock) (gives 0.3% final)
   • 1g (15) NH4Cl (if using M9-)

Tips

1. Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction.