PLASMID ISOLATION 

Protocol I

1. Spin 10 ml E.coli/plasmid suspension given in 50 ml Falcon tube at 3000rpm for 10 minutes.

2. Carefully pour off supernatant without disturbing the cell pellet.

Invert the tube and let it stand for 1 minute.

3. Add 200ul of glucose/Tris/EDTA to the pellet and gently resuspend the pellet.

4. Transfer the entire suspension to a microfuge tube.

5. Add 400ul of SDS/NaOH. Cap the tube and mix it very well.

6. Add 300ul of KoAc. Mix well and keep on ice for 5 minutes.

7. Spin tubes in microfuge at 12000rpm for 5-10 minutes. This pellets the proteins and chromosomal DNA along the side of the tube.

8. Transfer supernatant into another microfuge tube. Add equal volume of isopropanol. Close cap and mix well by rapidly inverting the tubes.

9. Leave at room temperature for 5 minutes.

10. Spin tubes at 12000rpm for 15 minutes. Pour off supernatant carefully.

White pellet at the bottom of the tube contains plasmid DNA and RNA.

11. Add 700ul 70% ethanol and wash the pellet by microfuging at 12000

rpm for 15 minutes.

12. Vacuum dry the pellets carefully.

13. Resuspend the pellet in 100ul TE pH8.0.

 

14. Take 10ul of the above plasmid add 2ulRNAase dye and incubate at 37⁰C for 10 minutes.

 

15. Load entire sample in 0.8% agarose gel and eletrophorese at 75 V for 1 hr.

16. Stain in dilute ethidium bromide solution for ten minutes, then photograph with U.V. transillumination.

If you complete the above protocol, go on to restriction digestion.

Protocol II.

Production of a Cleared Lysate

(This is the protocol you followed.)

1.  Pellet 1-3ml of saturated E. coli culture containing plasmid pPDS3 by centrifugation for 1-2 minutes at 10,000 x g in a microcentrifuge.
Pour off the supernatant and blot the tube upside-down on a paper towel to remove excess media.
2.  Completely resuspend the cell pellet in 250µl of Cell Resuspension Solution. Transfer the cells to a 1.5ml microcentrifuge tube if necessary.
3.Add 250µl of Cell Lysis Solution and mix by inverting the tube 4 times. The cell suspension should clear immediately.
[4.Add 10µl of Alkaline Protease Solution and mix by inverting the tube 4 times (DO NOT VORTEX). (We did not do this step.)]
5.Add 350µl of Neutralization Solution and mix by inverting the tube 4 times.

6. Centrifuge the lysate at 10,000 x g in a microcentrifuge for 5 minutes. If a pellet has not formed by the end of the centrifugation, centrifuge an additional 15 minutes.

 

Plasmid Purification Using Spin protocol

(This is the protocol that will be used in the class.

a.  Transfer the cleared lysate, approximately 850µl, by decanting into the Wizard Plus SV minipreps Spin Column inserted into a 2ml collection tube.

b.  Centrifuge the cleared lysate at 14,000 x g in a microfuge for 1 minute at room temperature.  Remove the spin column from the collection tube and discard the flow-through from the collection tube.

c.  Add 750µl of Column Wash solution to the spin column. Centrifuge at 14,000 x g for 1 minute at room temperature.  Remove the spin column from the collection tube and discard the flow-through from the collection tube.

d.  Add 250µl Column Wash solution to the spin column. Centrifuge at 14,000 x g for 2 minutes at room temperature. 

e.  Transfer the spin column to a clean, sterile 1.5 ml microcentrifuge tube.

f.  Elute the plasmid DNA by adding 50µl of Nuclease-free water to the spin column.  Centrifuge at 14,000 x g for 1 minute at room temperature.  Label the plasmid with the date, plasmid name and the group Number.

If you complete the above protocol, go on to restriction digestion.

Protocol III.

Plasmid Purification Using a Vacuum Manifold


Multiple Wizard® Plus Minipreps can be easily processed simultaneously with Promega's Vac-Man® or Vac-Man® Jr. Laboratory Vacuum Manifold. For each miniprep, prepare one Wizard® Miniprep Column. Attach one of the Syringe Barrels to the Luer-Lok® extension of each Minicolumn. Insert the tip of the Minicolumn/Syringe Barrel assembly into the vacuum manifold.
When all the columns are prepared, close all of the stopcocks.

Note: Thoroughly mix the Wizard® Minipreps DNA Purification Resin before removing
an aliquot. If crystals or aggregates are present, dissolve by warming the resin to 25-37°C
for 10 minutes. The resin itself is insoluble. Cool to 30°C before use.

Purifying the Plasmid.

1.Pipette 1ml of the resuspended resin into each barrel of the Minicolumn/Syringe assembly.
2.Carefully remove all of the cleared lysate from each miniprep (supernatant).
and transfer it to the barrel of the Minicolumn/Syringe assembly containing the resin.

Open the stopcocks and apply a vacuum to pull the resin/lysate mix into the Minicolumn.
When all of the sample has completely passed through the column, break the vacuum at the source.
3.Add 2ml of the Column Wash Solution (after the addition of ethanol) to the Syringe Barrel and reapply the vacuum to draw the solution through the Minicolumn.
4.Dry the resin by continuing to draw a vacuum for 30 seconds after the solution has been pulled through the column. Do not dry the resin for more than 30 seconds. Remove the
Syringe Barrel and transfer the Minicolumn to a 1.5ml microcentrifuge tube.

Centrifuge the Minicolumn at 10,000 x g in a microcentrifuge for 2 minutes to remove any residual Column Wash Solution.

5.Transfer the Minicolumn to a new microcentrifuge tube. Apply 50µl of water to the Minicolumn and wait 1 minute. Centrifuge the tube at 10,000 x g in a microcentrifuge for 20 seconds to elute the DNA.
6.Remove and discard the Minicolumn. Store the plasmid DNA in the microcentrifuge tube at 4°C or -20°C.

Restriction digestion

Once the plasmid is purified, it will be digested using the same method that we used to digest lambda  DNA.  Digest for 2 hours at 37°C and store the samples in -20°C till the next lab.