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30/07/13
From 2013.igem.org
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| + | <div style="font-family:arial;padding:5px;border-radius:5px;border:5px solid #FF2800;"> | ||
| + | {| style="color:#87EA00;background-color:#FFFFFF;" cellpadding="2" cellspacing="2" border="0" bordercolor="#000000" width="100%" align="center" | ||
| + | !align="center"|[[Team:Leicester|Home]] | ||
| + | !align="center"|[[Team:Leicester/Team|Team]] | ||
| + | !align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile] | ||
| + | !align="center"|[[Team:Leicester/Project|Project]] | ||
| + | !align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]] | ||
| + | !align="center"|[[Team:Leicester/Modeling|Modeling]] | ||
| + | !align="center"|[[Team:Leicester/Notebook|Notebook]] | ||
| + | !align="center"|[[Team:Leicester/Safety|Safety]] | ||
| + | !align="center"|[[Team:Leicester/Attributions|Attributions]] | ||
| + | |} | ||
| + | </div> | ||
| + | |||
| + | <p> | ||
| + | |||
| + | |||
==Transformation of the limonene Biobrick (BBa_K118025)== | ==Transformation of the limonene Biobrick (BBa_K118025)== | ||
Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI) | Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI) | ||
| + | |||
| + | <b>Protocol:</b> | ||
| + | *Start thawing the competent cells on ice | ||
| + | *Seperate 4 pre-chilled eppendorf tubes; | ||
| + | **#Resistance/Viability test | ||
| + | **#Ligation experiment with 10ul DNA | ||
| + | **#Ligation experiment with 5ul DNA | ||
| + | **#Positive control using 1ul RFP | ||
| + | *Add 50ul of thawed competent cells and then the aforementioned volume of DNA to each eppendorf tube. | ||
| + | *For each eppendorf tube, pipette the solution gently to mix. Ensure the cells are kept on ice. | ||
| + | *Close the tubes and incubate the cells on ice for 30 minutes | ||
| + | *Heat shock the cells by immersion in a pre-heated water bath at 42'C for 60 seconds | ||
| + | *Incubate the cells on ice for 5 minutes | ||
| + | *Add the 200ul of SOC (SOB + 0.4g glucose) media to each tube | ||
| + | *Incubate the cells at 37'C for 2 hours | ||
| + | *Plate onto petri dishes (ensure your petri dishes are labelled correctly): | ||
| + | **#For eppendorf 1, plate 100ul onto a chloroamphenicol petri dish and another 100ul of the solution onto a LB (luria broth) petri dish | ||
| + | **#For eppendorf 2, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes | ||
| + | **#For eppendorf 3, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes | ||
| + | **#For eppendorf 4, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes | ||
| + | *Incubate the plates at 37'C overnight | ||
Latest revision as of 14:37, 2 August 2013
| Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
|---|
Transformation of the limonene Biobrick (BBa_K118025)
Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI)
Protocol:
- Start thawing the competent cells on ice
- Seperate 4 pre-chilled eppendorf tubes;
- Resistance/Viability test
- Ligation experiment with 10ul DNA
- Ligation experiment with 5ul DNA
- Positive control using 1ul RFP
- Add 50ul of thawed competent cells and then the aforementioned volume of DNA to each eppendorf tube.
- For each eppendorf tube, pipette the solution gently to mix. Ensure the cells are kept on ice.
- Close the tubes and incubate the cells on ice for 30 minutes
- Heat shock the cells by immersion in a pre-heated water bath at 42'C for 60 seconds
- Incubate the cells on ice for 5 minutes
- Add the 200ul of SOC (SOB + 0.4g glucose) media to each tube
- Incubate the cells at 37'C for 2 hours
- Plate onto petri dishes (ensure your petri dishes are labelled correctly):
- For eppendorf 1, plate 100ul onto a chloroamphenicol petri dish and another 100ul of the solution onto a LB (luria broth) petri dish
- For eppendorf 2, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
- For eppendorf 3, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
- For eppendorf 4, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
- Incubate the plates at 37'C overnight
