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
Following the directions from the Qiagen manual for midi-preps.
- Resuspend bacterial pellet in 4mL of Buffer P1 by pipetting up and down
- Add 4mL of Buffer P2 (lysis buffer), mix by inverting sealed tube 4-6 times. (Note: solution turns blue)
- 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)
- Centrifuge at ≥ 20,000xg for 30 minutes at 4C
- Transfer supernatant to new falcon tube and mix the sample again once it's in the new tube.
- Centrifuge at ≥ 20,000xg for 15 minutes at 4C
- Equilabrate a Qiagen tip-100 by applying 4mL of Buffer QBT. Place tip in holder and rest over empty falcon tube to collect.
- Pour supernatant from centrifuged tube onto column and allow to flow over column (This binds the DNA to the column)
- Wash column with 2x10mL of Buffer QC
- Elute DNA with 5mL of Buffer QF
- Divide DNA among 6 eppendorf tubes
- Add 0.583mL of isopropanol to each eppendorf tube to precipitate the DNA
- Centrifuge at ≥ 15,000xg for 30minutes at 4C
- Decant the supernatant from the pellet (we could not see the DNA pellet)
- 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)
- Centrifuge at ≥ 15,000g for 10 minutes at 4C
- Carefully discard supernatant
- Air dry the pellet
- Resuspend the pellet in TE buffer, pH 8.0
After going through this procedure, I quantified the DNA using the UV/Vis.
- Take a blank spectrum - 198uL of water
- Take a sample spectrum - 2uL DNA added to the 198uL of water and mixed.
- Quantify DNA concentration: Concentration = A(260)*50μg/mL*DilutionFactor
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.
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