IGEM:IMPERIAL/2006/Protocols/mini prep of culture

Miniprep of cultures

• Transfer 1.5ml of culture into eppendorf tubes.
• Spin for 20seconds and remove supernatant.
• Take out Solution I and III from the fridge with syringes. Solution II (if it is cold it precipitates) is on the bench. (use yellow pipette for syringes)
• Add 100ul of solution I and resuspend the pellet by vortexing.
• Add 200ul of solution II.
• Add 150ul of solution III.
• Mix them well by inverting the tubes. White precipitates appears.
• Spin for 3min. In the meantime, label tubes.
• Pour the supernatants in to the new labelled tubes, add 1ml of ethanol and mix.
• Spin for another 3min and remove supernatant.
• Spin to remove residual liquid and then resuspend in 50ul of MiliQ (1 sec. vortex. Don’t resuspend the pellet).
• NB: If the culture was grown during the day (8hrs), then resuspend in 25μL to obtain approximately the same concentration.
• Spin for 1min.
• Ready to digest.

Solution 1

• 90g glucose
• 10ml 0.5M EDTA
• 6.25ml 2M Tris pH8
• make upto 500ml

Solution 2

• For 500 mls
• 50ml 10%SDS
• 20ml NaOH

Solution 3

• 147g potassium acetate
• 57.5ml glacial acetic acid
• make upto 500ml