Glasgow/Wetlab/Protocols
From 2007.igem.org
(Difference between revisions)
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== Transforming BioBricks == | == Transforming BioBricks == | ||
| - | #Split the competent ''E. coli'' cells (~ | + | #Split the competent ''E. coli'' cells (~75 µl) in two. |
| - | #Competent cells (~ | + | #Competent cells (~33 µl) into eppendorfs on ice. |
#Add 1µl of plasmid DNA. | #Add 1µl of plasmid DNA. | ||
#Incubate on ice for at least 5 mins. | #Incubate on ice for at least 5 mins. | ||
| - | #Heat shock + | + | #Heat shock +42ºC for 45 secs. |
#Back on ice for 2 mins to reduce damage. | #Back on ice for 2 mins to reduce damage. | ||
#Add 1ml of soc media. | #Add 1ml of soc media. | ||
#Incubate +37ºC in a shaker for 1 hour. | #Incubate +37ºC in a shaker for 1 hour. | ||
#Spread the reaction on kan/carb LB plates. | #Spread the reaction on kan/carb LB plates. | ||
| - | #Grow + | + | #Grow +37ºC overnight. |
== Minimal Media and Trace Elements == | == Minimal Media and Trace Elements == | ||
=== Trace Elements === | === Trace Elements === | ||
| - | #In | + | #In 800 ml dH2O dissolve 5 g Na2 EDTA and convert to pH7. |
#Add the following in order, correcting to pH7 after each. | #Add the following in order, correcting to pH7 after each. | ||
| - | #*FeCl3 (.H2O) 0.5 (0. | + | #*FeCl3 (.H2O) 0.5 (0.83 g) |
| - | #*ZnCl2 0. | + | #*ZnCl2 0.05 g |
| - | #*CuCl2 0. | + | #*CuCl2 0.01 g |
| - | #*CoCl2.6H20 0. | + | #*CoCl2.6H20 0.01 g |
| - | #*H3BO3 0. | + | #*H3BO3 0.01 g |
| - | #*MnCl2.6H2O (.4H2O) 1. | + | #*MnCl2.6H2O (.4H2O) 1.6 g (1.35 g) |
#Make up to 1 litre, autoclave and store at 4 °C. | #Make up to 1 litre, autoclave and store at 4 °C. | ||
=== M9 === | === M9 === | ||
#Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds | #Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds | ||
| - | #* | + | #*6 g Na2HPO4 |
| - | #* | + | #*3 g KH2PO4 |
| - | #*0. | + | #*0.5 g NaCl |
#The add | #The add | ||
| - | #* | + | #*1 ml 1M MgSO4 |
| - | #* | + | #*200 µl 1M CaCl2 |
#*1 ml Thiamine (40mg ml-1 stock) | #*1 ml Thiamine (40mg ml-1 stock) | ||
#*10 ml Trace Elements | #*10 ml Trace Elements | ||
#Also add as necessary | #Also add as necessary | ||
| - | #* | + | #*15 ml (13C-) Glucose (20% stock) (gives 0.3% final) |
| - | #* | + | #*1 g (15) NH4Cl (if using M9-) |
=== Tips === | === Tips === | ||
#Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction. | #Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction. | ||
| + | |||
| + | == Induction of Luciferase by Toluene and its Derivatives == | ||
| + | '''(adapted from Willardson et al., 1998)''' | ||
| + | |||
| + | #Grow single colonies of DH5α cells containing pGLTUR in 10 ml LB medium containing 100 µM carbenicillin at 37ºC until OD600 is between 0.2 and 1.0. | ||
| + | #Dilute to an OD600 of 0.2 with LB medium. | ||
| + | #Induce luciferase transcription by mixing 0.9 ml of the diluted culture with 0.9 ml of LB medium containing increasing amounts of toluene or its derivative compounds. | ||
| + | #Incubate in 2.0 ml glass vials (sealed with Teflon septa to avoid loss of the volatile organic compound) at 37ºC for 30 min and then cool on ice for 10 min. | ||
| + | #Measure luciferase activity | ||
| + | #75 µl of the induced culture is lysed by addition of 25 µl of 4x lysis buffer (100 mM Tris.HCl [pH 7.8], 32 mM NaH2PO4, 8mM dithiothreitol, 8 mM CDTA, 4% [vol/vol} Triton-X 100, and 200 µg of polymyxin B sulphate per ml). | ||
| + | #25 µl of the lysed cell solution was added to 25 µl of a combined 4x concentrate of luciferase substrates A and B (Analytical Luminescence Laboratories) to give a final 2x concentration of each substrate. | ||
| + | #Luminesence read immediately after substrate addition for 45 s in a Turner TD-20e luminometer. | ||
Revision as of 08:22, 20 July 2007
Contents |
Transforming BioBricks
- Split the competent E. coli cells (~75 µl) in two.
- Competent cells (~33 µl) into eppendorfs on ice.
- Add 1µl of plasmid DNA.
- Incubate on ice for at least 5 mins.
- Heat shock +42ºC for 45 secs.
- Back on ice for 2 mins to reduce damage.
- Add 1ml of soc media.
- Incubate +37ºC in a shaker for 1 hour.
- Spread the reaction on kan/carb LB plates.
- Grow +37ºC overnight.
Minimal Media and Trace Elements
Trace Elements
- In 800 ml dH2O dissolve 5 g Na2 EDTA and convert to pH7.
- Add the following in order, correcting to pH7 after each.
- FeCl3 (.H2O) 0.5 (0.83 g)
- ZnCl2 0.05 g
- CuCl2 0.01 g
- CoCl2.6H20 0.01 g
- H3BO3 0.01 g
- MnCl2.6H2O (.4H2O) 1.6 g (1.35 g)
- 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
- 6 g Na2HPO4
- 3 g KH2PO4
- 0.5 g NaCl
- The add
- 1 ml 1M MgSO4
- 200 µl 1M CaCl2
- 1 ml Thiamine (40mg ml-1 stock)
- 10 ml Trace Elements
- Also add as necessary
- 15 ml (13C-) Glucose (20% stock) (gives 0.3% final)
- 1 g (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.
Induction of Luciferase by Toluene and its Derivatives
(adapted from Willardson et al., 1998)
- Grow single colonies of DH5α cells containing pGLTUR in 10 ml LB medium containing 100 µM carbenicillin at 37ºC until OD600 is between 0.2 and 1.0.
- Dilute to an OD600 of 0.2 with LB medium.
- Induce luciferase transcription by mixing 0.9 ml of the diluted culture with 0.9 ml of LB medium containing increasing amounts of toluene or its derivative compounds.
- Incubate in 2.0 ml glass vials (sealed with Teflon septa to avoid loss of the volatile organic compound) at 37ºC for 30 min and then cool on ice for 10 min.
- Measure luciferase activity
- 75 µl of the induced culture is lysed by addition of 25 µl of 4x lysis buffer (100 mM Tris.HCl [pH 7.8], 32 mM NaH2PO4, 8mM dithiothreitol, 8 mM CDTA, 4% [vol/vol} Triton-X 100, and 200 µg of polymyxin B sulphate per ml).
- 25 µl of the lysed cell solution was added to 25 µl of a combined 4x concentrate of luciferase substrates A and B (Analytical Luminescence Laboratories) to give a final 2x concentration of each substrate.
- Luminesence read immediately after substrate addition for 45 s in a Turner TD-20e luminometer.
