Stanford/BIOE44:Module 1:Day1

M1: Day 1 - Plasmid Extraction and Restriction Digest

Introduction

• plasmids
• mini preps - purpose of each step
• parts
• restriction enzymes
  • components of a digest

Before Class

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In Class

Part 1: Plasmid extraction (aka miniprep)

At the beginning of class you will be given a 5mL culture of E. coli harboring a plasmid that contains a BioBrick part. We will extract the plasmid from the bacteria using a kit (QIAPrep Spin Miniprep kit). Then we'll measure the concentration of the plasmid DNA extracted using a nanodrop spectrophotometer.

Mini-Prep Protocol

Collect the Cells

1. Label three microcentrifuge tubes.
2. Pour approx. 1.5 mL of culture into each tube.
3. Centrifuge at 10000xg for 5 minutes.
4. Aspirate off supernatant. Be care to not suck up your cell pellet.

Break the cells open

1. Resuspend the cells in tube #1 with 250uL of Buffer P1.
2. Transfer all liquid from tube #1 to tube #2 and resuspend the pellet.
3. Repeat from tube #2 to tube #3. (Should end up with all the cells in tube #3. Throw away tubes 1 and 2)
4. Add 250uL of Buffer P2 to cells. Invert the tube 4-6 times.

Part 2: Restriction Enzyme Digest

We will take the plasmid we just extracted and set up a restriction digest to cut the part out of the plasmid.

For Next Time

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