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Team:University College London/LabBook/Week3
From 2012.igem.org
Monday (18.6.12)
Aim – Day 1 of Generating Competent Cells: In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.
In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.
Method
Step 1 – Setting up Plates: Pour several plates with minimal agar.
Step 2 – Streaking: Streak the chosen cell line onto the minimal agar plate.
Step 3 – Incubation: Incubate at 37oC overnight
Tuesday (19.6.12)
Aim – Picking Colonies: Today we check the colony formation from yesterday’s culture, and pick a colony for inoculation
Method
Step 1 -Creating culture media: In a sterile environment, set up a falcons, with 5mls of Lysogeny Broth and 100ul 1M MgS04.
Step 2 – 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 3 - Cell culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.
Wednesday (20.6.12)
Aim – Permeabilisation: Incubating Cells in the presences of CaCl2 will prepare the cell wall, such that it becomes permeable to DNA.
Method
Step 1 – Innoculation: Inoculate 100mls of Lysogeny Broth in pre-warmed conical with 1ml of the overnight culture from Day 2
Step 2 – Incubation: Incubate for two hours in a 37oC shaker until the cells are at the early log phase of the growth curve. Measure absorbance on a spectrometer until A6000 is approximately 0.3.
Step 3 – Incubation: Transfer to a chilled sterile 50ml Falcon tube and incubate on ice for 10 minutes Step 4 – Centrifuge:
-Time: 5 mins -G – 3300 -Temperature: 180C
Step 5 – Incubate: Resus in 10ml of ice cold 0.1M CaCl2 in 15% glycerol and incubate on ice for 30 minutes.
Step 6 – Centrifuge: -Time: 5 mins -G – 3300 -Temperature: 180C
Step 7 – Transfer: Resus in 1ml of ice cold 0.1M CaCl2 in 15% glycerol. Transfer 100ul aliquots to pre-chilled, pre-labelled eppendorf tubes. Store at -70oC
Step 8 – Centrifuge -Time: 5 mins -G – 3300 -Temperature: 180C
Step 9 - Storage: Store cells at -80oC
Step 1 -Creating culture media: In a sterile environment, set up a falcons, with 5mls of Lysogeny Broth and 100ul 1M MgS04.
Step 2 – 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 3 - Cell culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.
