User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2012/09/11: Difference between revisions
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==Description== | ==Description== | ||
# Add | 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 | |||
==Data== | ==Data== |
Revision as of 07:08, 12 September 2012
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ObjectiveExtract 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 DescriptionFollowing the directions from the Qiagen manual for midi-preps.
After going through this procedure, I quantified the DNA using the UV/Vis.
Data
NotesThis area is for any observations or conclusions that you would like to note.
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