Stanford/BIOE44:Module 1:Day1

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M1: Day 1 - Plasmid Extraction and Restriction Digest

Introduction

  • overview of what we are going to do
  • standard assembly
    • plasmids
    • parts
    • promoters
  • methods stuff:
    • mini preps - purpose of each step
    • restriction enzymes
    • components of a digest

Extra Reading/References

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

Materials

  • 5mL E. coli culture
  • microcentrifuge tubes
  • Buffers P1, P2, N3, and PE
  • Qiaprep column
  • sterile water

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 the proteins are denatured)
  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 proteins 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. Discard the flow through.
  3. Apply 750uL of Buffer PE and centrifuge for 1 minute at max speed.
  4. Discard the flow through. Centrifuge the column for an additional 1 minute to remove residual wash buffer.
  5. Place the column in a clean 1.5ml micro-centrifuge tube. Apply 50uL sterile water to the column. Let it stand for 1 minute. Centrifuge at max speed for 1 minute.

'Measure the concentration

  1. Measure the concentration of your newly prepped plasmid with the nano-drop. Record the concentration and 260/280 in your lab notebook.

Part 2: Restriction Enzyme Digest

We will take the plasmid we just extracted and set up a restriction digest to cut out the color generator genes.

For Next Time

  • Please read:
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