User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2012/09/11

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Objective

Extract plasmid DNA from the midi-prep that Stefano performed. This plasmid contains the gene for adenosine deaminase. The information for the gene can be found here

Description

Following the directions from the Qiagen manual for midi-preps.

  1. Resuspend bacterial pellet in 4mL of Buffer P1 by pipetting up and down
  2. Add 4mL of Buffer P2 (lysis buffer), mix by inverting sealed tube 4-6 times. (Note: solution turns blue)
  3. Add 4mL of chilled Buffer P3 (precipitation buffer), mix by inverting sealed tube 4-6 times. (Note: blue color goes away and lipids precipitate) (Note: Stefano performed the first three steps)
  4. Centrifuge at ≥ 20,000xg for 30 minutes at 4C
  5. Transfer supernatant to new falcon tube and mix the sample again once it's in the new tube.
  6. Centrifuge at ≥ 20,000xg for 15 minutes at 4C
  7. Equilabrate a Qiagen tip-100 by applying 4mL of Buffer QBT. Place tip in holder and rest over empty falcon tube to collect.
  8. Pour supernatant from centrifuged tube onto column and allow to flow over column (This binds the DNA to the column)
  9. Wash column with 2x10mL of Buffer QC
  10. Elute DNA with 5mL of Buffer QF
  11. Divide DNA among 6 eppendorf tubes
  12. Add 0.583mL of isopropanol to each eppendorf tube to precipitate the DNA
  13. Centrifuge at ≥ 15,000xg for 30minutes at 4C
  14. Decant the supernatant from the pellet (we could not see the DNA pellet)
  15. Resuspend pellet in 1mL of 70% ethanol (i.e. add 1mL of 70% ethanol to the first tube, pipette up and down and transfer contents to next tube, repeat until finished.) (Note: at this point we could observe the DNA as a clearish-white solid in solution)
  16. Centrifuge at ≥ 15,000g for 10 minutes at 4C
  17. Carefully discard supernatant
  18. Air dry the pellet
  19. Resuspend the pellet in TE buffer, pH 8.0

After going through this procedure, I quantified the DNA using the UV/Vis.

  1. Take a blank spectrum - 198uL of water
  2. Take a sample spectrum - 2uL DNA added to the 198uL of water and mixed.
  3. Quantify DNA concentration: Concentration = A(260)*50μg/mL*DilutionFactor

Data

DNA concentration = 0.025*50μg/mL*100 = 125μg/mL

The Solution was labeled with the concentration and the name of the gene and placed in Dr. Hartings' freezer.

Notes

This area is for any observations or conclusions that you would like to note.


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