User:Kfifer
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Lab Notebook
June 14, 2006
Plasmid Miniprep Samples
- lac operon promoter
- R0010-1
- R0010-2
- promoter and GFP
- E0241-1
- E0241-2
- GFP
- E7104-1
- E7104-2
Protocol
- Remove growing LB cultures from warm shaker.
- Set aside 1mL of each sample for later glycerol storage at -80 if the transformation worked.
- Use an eppendorf tube to spin down the rest of the LB culture (probably in about 3 spins each - keep the pellet each time and remove the supernatant by just pouring it off. The final time, pipet the supernatant away so that there's nothing left but pellet.)
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. Pipet up and down. No cell clumps should be visible after resuspension of the pellet.
- Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
