Protocol for Alkaline Lysis Miniprep

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Lab Safety - please contact your institution's health and safety office prior to implementing the laboratory procedure outlined. Comply with all laboratory safety regulations in accordance with your institution's health and safety office.


Materials and Reagents
 Reagents:  Materials: 
  • Resuspension Buffer R3 – 50 mM Tris-HCl, pH 8.0; 10 mM EDTA 
  • RNase A – 20 mg/mL in Resuspension Buffer R3
  • Lysis Buffer L7 – 200 mM NaOH, 1% w/v SDS 
  • Precipitation Buffer N4
  • Wash Buffer W9, ethanol already added 
  • E. coli DH5α cells harboring pET26b-folA
  • Micro-centrifuge 
  • 1.5 mL sterile micro-centrifuge tubes
  • Spin columns
  • Heat block

1. Step 1 will be done for you: Inoculate LB-medium with a single colony. Grow to saturation (overnight).

2. Write down the protocol from the PureLink® Quick Plasmid Miniprep Kit manual prior to coming to the lab. Make sure you include the safety considerations when working with the buffers specified in the manual. You will be using a centrifuge to purify your plasmids. Each student will miniprep 1 reaction. Each student will pellet 1.4 mL of bacterial culture/miniprep. Also, please note that the final elution step will change. Instead of 75 µL of TE buffer, you will add 40 µL of nuclease free water.

Once you are finished please wash your hands!


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BBS OER Lab Manual Copyright © 2021 by Felicia Vulcu/ Vivian Leong/ Caitlin Mullarkey (Department of Biochemistry and Biomedical Sciences McMaster University) is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.

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