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. Solution should become slightly clear and viscous. (This is the step where the cells are lysed and all the goo inside the cells comes out)
5. Add 350uL of Buffer N3 to the solution. Mix immediately by inverting the tube 4-6 times. (Thick white goo will form - this is all the cellular junk precipitating out)
6. Centrifuge for 10 minutes at max speed (approx 17000xg). While centrifuging, take out a column and label it.

Isolate the plasmids

1. Apply the supernatant from the tube to the column by pipetting (approx 800ul). Be care to not pipette any of the white stuff - this will clog your column and reduce your yield.
2. Centrifuge for 1 minute at max speed.

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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