DNA isolation, restriction, visualitation, and quantification
Important points on DNA isolation
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Transcript of Important points on DNA isolation
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Important points on DNA isolation
This is not the entire protocol but just some important points
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Miniprep
• After picking colonies from the transformations, we will let these cultures grow overnight
• We will use a miniprep kit to isolate DNA from the cultures
• We need to add Ampicillin when we grow the cultures inorder to keep the plasmid
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Do we need to add Arabinose?
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To isolate plasmid DNA
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Protocol
• Spin the cultures to get the bacterial pellet
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Protocol• Add resuspension solution
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Protocol
• Vortex or pipet vigorously to resuspend the bacterial cells.
• This is the only time during this protocol where you can use the vortex.
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Protocol• Add 250 mL of the cell lysis solution and mix thoroughly
by inverting the tube 6-8 times.
• Do not vortex because you do not want to shear the DNA.
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Protocol
• Add 350 mL of neutralization solution
• Mix immediately and gently but do not vortex.
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After adding the cell lysis solution and neutralization solution this is what you should
seethe tube on the left isbefore centrifugation andthe tube on the right isafter centrifugation
save the supernatantThat is where the plasmidDNA isThe white stuff containsproteins and chromosomalDNA
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Protocol
• Centrifuge for 5 minutes at top speed to pellet cell debris and chromosomal DNA.
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Remove supernatant carefully
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Protocol• Transfer the supernatant to the supplied spin column.
• Do NOT put the sample in the supplied collection tube
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Protocol
• Avoid disturbing the white precipitate when you transfer the supernatant.
• You should now setup your tubes like the picture on the right– The column is inside the collection tube
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Protocol
• Centrifuge for 1 minute at top speed and discard the flow through. That is what ends up in the collection tube.
• Put the column back into the same collection tube.
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Protocol• Add the wash solution
• Centrifuge and discard the flow through
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Protocol
• Discard the flow through
• Centrifuge for 1 minute at 3000 RPM to get rid of any residual ethanol from the wash solution
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Protocol• Transfer the column to a new tube
• Add the elution buffer
• Centrifuge and SAVE the DNA
• That is what is now in the tube