Glasgow/Wetlab/Protocols

From 2007.igem.org

(Difference between revisions)
(Transforming BioBricks)
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

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.