Important points on DNA isolation

1
Important points on DNA isolation
2
Plasmid isolation
3
Isolation of DNA miniprep

• Bacterial cells are grown overnight to stationary
  phase
• Cells are collected by centrifugation
• Cells resuspended in a resuspension buffer
• Add lysis solution which contains SDS and NaOH
• SDS is a detergent and dissolves the phospholipid
  and protein components of the cell membrane cell
  contents released
• NaOH denatures plasmid and chromosomal DNA into
  single strands but single stranded plasmid loops
  remain linked together (important in separating
  plasmid and chromosomal DNA)
• Add a neutralization solution
• Potassium acetate and acetic acid forms an
  insoluble precipitate of SDS/lipid/protein and
  neutralizes the NaOH
• At neutral pH, the DNA renatures
• Centrifugation chromosomal DNA in the pellet,
  plasmid DNA in the supernatant
• Add to a wash column
• Centrifuge and wash with ethanol plasmid DNA is
  now in the pellet
• Resuspend DNA

4
Bacterial cells

• After your colonies have grown, you (or some
  designate) will pick 2 colonies from the pBR322
  plate and 2 colonies from the pUC18/19 plate and
  grow cultures from them.
• You will use a miniprep kit to isolate DNA from
  the cultures

5
Protocol

• For the pUC 18/19 plasmid, you need 5ml of the
  bacterial culture.
• For the pBR322 plasmid, you need 10 ml of the
  bacterial culture.
• Spin these cultures at 12,000 RPM for 10 minutes
  in the centrifuge at the back of the room. Make
  sure the tubes are balanced.

6
In this rotor, 15 ml tubes go in one spot and 50
ml tubes go in another
7
Notice the correct placement of tubes in this
rotor
8
Protocol

• Add 250 mL of the resuspension solution and
  transfer the solution to a microcentrifuge tube.
• What is RNase and why do you want it present when
  isolating DNA?

9
Protocol

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

10
Protocol

• Add 250 mL of the cell lysis solution and mix
  thoroughly by inverting the tube 4-6 times.
• Do not vortex because you do not want to shear
  the DNA. Do not incubate for more than 5 minutes
  because you do not want to denature the DNA.

11
Protocol

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

12
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
13
Protocol

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

14
Remove supernatant carefully
15
Protocol

• Transfer the supernatant to the supplied spin
  column.

• Do NOT put the sample in
• the supplied collection tube

16
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

17
Protocol

• Centrifuge for 1 minute and discard the flow
  through. That is what ends up in the collection
  tube.
• Put the column back into the same collection
  tube.

18
Protocol

• Add 500 mL of the wash solution
• Centrifuge for 30-60 seconds and discard the flow
  through
• Repeat the wash procedure using another
• 500 mL of the wash solution

19
Protocol

• Discard the flow through
• Centrifuge for 1 minute to get rid of any
  residual ethanol from the wash solution

20
Protocol

• Transfer the column to a new tube
• Add 50 mL of the elution buffer
• Incubate for 2 minutes
• Centrifuge for 2 minutes and SAVE the DNA
• That is what is now in the tube