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Team:University College London/LabBook/Week9
From 2012.igem.org
Contents |
Monday 6.7.12
Aim: Repeat the inoculation of the ligation products (J23119 + B0034) done last week. Innoculation is only done for the (J23119 + B0034), but not for the (starvation promoter + B0034) because that ligation did not transform.
Method
Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.
(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)
Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube
Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.
Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.
| Ligation | No. innoculations | Broth | Antibiotic |
|---|---|---|---|
| J23119 + B0034 | 3 |
Tuesday 7
Aim: Check results of Colony Picking
Results: The table below indicates whether there was growth for the BioBricks ligations
| Ligation | Growth/No Growth |
|---|---|
| J23119+B0034 sample 1 | Growth |
| J23119+B0034 sample 2 | Growth |
| J23119+B0034 sample 3 | Growth |
Conclusion: We can proceed now to miniprep the samples
Method:
Step 1 - Pellet Cells: Pellet 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000
Time = 2 min
Temperature = (15-25oC)
Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube.
Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min.
Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms.
Step 5 - Centrifuge:
g: 14000
Time:10 minutes
Temperature: (15-25oC)
Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge:
g = 10,000
Time: 1 minute
Temperature: (15-25oC)
Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 8 - Remove residual ethanol: Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 10 - Storage: Store DNA at 4oC or -20oC
Was this run on a gel?
Monday 6.7.12
Aim: Repeat the inoculation of the ligation products (J23119 + B0034) done last week. Innoculation is only done for the (J23119 + B0034), but not for the (starvation promoter + B0034) because that ligation did not transform.
Method
Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.
(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)
Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube
Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.
Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.
| Ligation | No. innoculations | Broth | Antibiotic |
|---|---|---|---|
| J23119 + B0034 | 3 |
Tuesday 7
Aim: Check results of Colony Picking
Results: The table below indicates whether there was growth for the BioBricks ligations
| Ligation | Growth/No Growth |
|---|---|
| J23119+B0034 sample 1 | Growth |
| J23119+B0034 sample 2 | Growth |
| J23119+B0034 sample 3 | Growth |
Conclusion: We can proceed now to miniprep the samples
Method:
Step 1 - Pellet Cells: Pellet 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000
Time = 2 min
Temperature = (15-25oC)
Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube.
Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min.
Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms.
Step 5 - Centrifuge:
g: 14000
Time:10 minutes
Temperature: (15-25oC)
Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge:
g = 10,000
Time: 1 minute
Temperature: (15-25oC)
Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 8 - Remove residual ethanol: Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge:
g - 10,000
Time - 1 minute
Temperature: (15-25oC)
Step 10 - Storage: Store DNA at 4oC or -20oC
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