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 (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

Do we need to add Arabinose?

To isolate plasmid DNA

Protocol

• Spin the cultures to get the bacterial pellet

Protocol

• Add resuspension solution

Protocol

• Vortex or pipet vigorously to resuspend the bacterial cells.

• This is the only time during this protocol where you can use the vortex.

Protocol

• Add 250 L 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

• Add 350 L of neutralization solution

• Mix immediately and gently but do not vortex.

After adding the cell lysis solution and neutralization solution this is what you should

see

the 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

Protocol

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

Remove supernatant carefully

Protocol

• Transfer the supernatant to the supplied spin column.

•Do NOT put the sample in the supplied collection tube

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

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.

Protocol

• Add the wash solution

• Centrifuge and discard the flow through

Protocol

• Discard the flow through

• Centrifuge for 1 minute at 3000 RPM to get rid of any residual ethanol from the wash solution

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