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 (S3 and pGLO)
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. Protocol
Add resuspension solution

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. 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.

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

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

14. 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

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

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. 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