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Important points on DNA isolation

Important points on DNA isolation. This is not the entire protocol but just some important points. 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

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Important points on DNA isolation

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  1. Important points on DNA isolation This is not the entire protocol but just some important points

  2. 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

  3. Do we need to add Arabinose?

  4. To isolate plasmid DNA

  5. Protocol • Spin the cultures to get the bacterial pellet

  6. Add resuspension solution Protocol

  7. Protocol • Vortex or pipet vigorously to resuspend the bacterial cells. • This is the only time during this protocol where you can use the vortex.

  8. 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. Protocol

  9. Add 350 mL of neutralization solution Mix immediately and gently but do not vortex. Protocol

  10. After adding the cell lysis solution and neutralization solution this is what you should see the tube on the left is before centrifugation and the tube on the right is after centrifugation save the supernatant That is where the plasmid DNA is The white stuff contains proteins and chromosomal DNA

  11. Protocol • Centrifuge for 5 minutes at top speed to pellet cell debris and chromosomal DNA.

  12. Remove supernatant carefully

  13. Transfer the supernatant to the supplied spin column. Protocol • Do NOT put the sample in • the supplied collection tube

  14. 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 Protocol

  15. 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.

  16. Add the wash solution Centrifuge and discard the flow through Protocol

  17. Protocol • Discard the flow through • Centrifuge for 1 minute at 3000 RPM to get rid of any residual ethanol from the wash solution

  18. Transfer the column to a new tube Add the elution buffer Centrifuge and SAVE the DNA That is what is now in the tube Protocol

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