Plasmid Mini-preparation of pGreen

 

 

Culture and Reagents                                                Supplies and Equipment

 

E. coli/pGreen overnight culture                                    100-1,000-ml micropipettor + tips

    (grown in 5ml LB/amp broth)                                    0.5 – 10 ml micropipettor + tips

Glucose/Tris/EDTA (GTE)                                           1.5 – ml tubes        

SDS/sodium hydroxide (SDS/NaOH)                 Hair dryer
Potassium acetate/acetic acid (KOAc/HoAc)                beaker for waste/used tips

Isopropanol                                                                  10% - bleach solution or disinfectant

95%ethanol                                                                  disposable gloves

Tris/EDTA (TE)                                                           test tube rack                                                                                        beaker of crushed ice 

                                                                                    Microfuge

                                                                                    Paper towels

                                                                                    Permanent marker

 

                                               

Growing the Plasmid (step 1)

 

Growing  cells for Plasmid MiniPrep  – 2-3 days in advance of miniprep procedure.

 

Take a sterile 50 mL conical tube,  put in 5 mL of sterile LB/amp broth.  Pick one green colony cell mass from the plate of transformants (LB/amp +), and inoculate into th e broth tube.  Pick a cell mass that is well separated from other colonies to avoid scraping up additional colonies incubate at 37 degrees C.  If you have a shaker, an overnight incubation is fine.  However, if incubating at 37 degrees C without shaking, you can prepare culture 2-3 days in advance, and then store in refrigerator till ready to use.  The cells settle at bottom of tube so shake the tube to resuspend the cells before beginning this procedure.

 

Isolate Plasmid DNA (step 2)

(50 minutes)

 

The instructions below are for making duplicate minipreps, which provide balance in the microfuge and insurance if a critical mistake is made.

 

1.      Shake the culture tube to resuspend the E. coli cells.

 

2.      Label two 1.5 ml tubes with your initials.  Transfer 1000 ml (l ml) of       E.coli/pGreen overnight suspension into each tube.

 

3.      Close the caps, and place the tubes in a balanced configuration in the microfuge rotor.  Spin for 1 minute to pellet cells.

 

4.      Pour off the supernatant from both tubes into a waste beaker for later disinfection.  Take care not to disturb the cell pellets.  Invert the tubes, and touch their mouths to a clean paper towel, to wick off as much as possible of the remaining supernatant.

 

 

5.      Add 100 ml of ice-cold GTE solution to each tube.  Resuspend the pellets by pipetting solution in and out several times.  Hold the tubes up to the light to check that the suspension is homogeneous and that no visible clumps of cells remain.

6.      Add 200 ml of room temperature SDS/NaOH solution to each tube.  Close the caps, and mix the solutions by rapidly inverting the tubes about five times.

 

7.      Stand the tubes on ice for 5 minutes.  The suspension will become relatively clear.

 

 

8.      Add 150μl of ice-cold KOAc/HoAc solution to each tube.  Close the caps, and mix the solutions by rapidly inverting the tubes about five times.  A white precipitate will immediately appear. 

 

9.      Stand the tubes on ice for 5 minutes.

 

 

10.  Place the tubes in a balanced configuration in the microfuge rotor, and spin them at 12,000 rpm for 5 minutes to pellet the precipitate.

 

11.  Transfer 400 ml of supernatant from each tube into two clean 1.5 – ml tubes.  Avoid pipetting the precipitate.  Wipe off any precipitate clinging to the outside of the tip before expelling the supernatant.  Discard old tubes containing precipitate.

 

 

12.  Add 400 ml of isopropanol to each tube of supernatant.  Close the caps, and mix the solution by rapidly inverting the tubes about five times.  Stand the tubes at room temperature for only 2 minutes.  Coordinate with other groups so microcentrifuge is available at the end of the 2 minutes!

 

13.  Place the tubes in a balanced configuration in a microfuge rotor, and spin them at 10,000 rpm in microfuge  for 5 minutes to pellet the nucleic acids.  Align the tubes in the rotor so that the cap hinges point outward.  The nucleic acid residue, visible or not, will collect at the bottom of the tube under the hinge, during centrifugation.

 

 

14.  Pour off the supernatant from both tubes.  Take care not to disturb the nucleic acid pellets.  Wick off as much as possible of the remaining alcohol on a paper towel.

 

15.  Add 200 ml of 95% ethanol to each tube, and close the caps.  Flick the tubes several times to wash the pellets.

 

STOP POINT

            Store the DNA in ethanol at –20◦ C until you are ready to continue.

 

16.  Place the tubes in a balanced configuration in a microfuge rotor, and spin for 2-3 minutes to recollect the nucleic acid pellets. (see #13)

 

17.  Pour off the supernatant from both tubes.  Take care not to disturb the nucleic acid pellets.   Wick off as much as possible of the remaining alcohol on a paper towel.

 

18.  Dry the nucleic acid pellets, using one of the following methods:

a.       Direct a stream of warm air from a hair dryer across the open ends of the tubes for about 3 minutes.   Do not blow the pellets out of the tubes.

OR

 

b.      Close the caps, and pulse the tubes in the microfuge to pool the remaining ethanol.  Carefully, use the micropipettor to draw off the ethanol.  Let the pellets air dry at room temperature for 10 Minutes.

 

19.  Be sure that the nucleic acid pellets are dry and that all the ethanol has evaporated before proceeding to step 20.  Hold each tube up to the light and confirm that no ethanol droplets remain and that the nucleic acid pellet, if visible, appears white and flaky. Sniff the mouth of the tube; if ethanol is still evaporating, an alcohol odor will be detected.

 

20.  Add 15μl of TE to each tube.  Resuspend the pellets by scraping them with a pipet tip and vigorously pipetting in and out. Rince down the side of the tube several times, concentrating on the area where the pellet should have formed during centrifugation (beneath the cap hinge).  Check that all DNA is dissolved and that no particles remain in the tip or on the side of the tube.

 

21.  Pool the two DNA/TE solutions into one 1.5 ml tube and label.

 

 

STOP POINT

            Freeze the DNA/TE solution at – 20◦ C until you are ready to continue.  Thaw before using.

 

22.  Take the time for proper cleanup.

a.       A. Segregate for proper disposal culture tubes, paper towels, and micropipettor tips that have come into contact with E.coli.

b.      Disinfect the overnight culture, pipet tips, and the supernatant from step 4 with a 10% bleach solution or disinfectant (such as Lysol).

c.       Wipe down the lab bench with soapy water, a 10% bleach solution , or disinfectant.

d.      Wash your hands before leaving the laboratory.

 

 

 

 

Results and Discussion

 

The minipreparatin is a simple and efficient procedure for isolating plamid DNA.  You should be familiar with the molecular and biochemical effects of each reagent used in the protocol:

·        Glucose/Tris/EDTA: Glucose functions to maintain osmotic pressure, while the Tris buffers the cells at pH8.0.  EDTA binds divalent cations in the lipid bilayer, thus weakening the cell envelope.  Folowing cell lysis, EDTA limits DNA degradation by binding MG++ ions that are a necessary cofactor for bacterial nucleases.

·        SDS/sodium hydroxide:  This alkaline mixture lyses the bacterial cells.  The detergent SDS dissolves the lipid components of the cell envelope and the cellular proteins.  Sodium hydroxide denatures the chromosomal and plasmid DNA into single strands; the intact circles of plasmid DNA remain intertwined.

·        Potassium acetate/acetic acid:  The acetic acid brings the pH to neutral, allowing the DNA strands to renature.  The large, disrupted chromosomal strands cannot rehybridize perfectly but instead collapse into a partially hybridized tangle.  At the same time, the Postassium acetate precipitates the SDS from the cell suspension, along with the associated proteins and lipids.  The renaturing chromosomal DNA is trapped in the SDS/lipid /protein precipitate.  Only smaller plasmid DNA, fragments of chromosomal DNA, and RNA molecules escape the precipitate and remain in solution.

·        Isopropanol:  The alcohol rapidly precipitates nucleic acids but precipitates proteins slowly.  Thus, a quick precipitation preferentially brings down nucleic acids.

·        Ethanol:  A wash with ethanol removes some remaining salts and SDS from the preparation

·        Tris-EDTA:  Tris buffers the DNA solution, EDTA protects the DNA from degradation by DNAses by binding the divalent cations (especially MG++) that are necessary cofactors for the DNAse activity.

 

 

 

 

Questions

 

 

1.      Consider the three major classes of biologically important molecules:  proteins, lipids, and nucleic acids.  Which steps of the miniprep procedure act on proteins?  On lipids? On nucleic acids?

 

2.      Which aspect of plamid DNA structure allows it to renature efficiently in step 8?

 

 

3.      What other kinds of molecules, in addition to plasmid DNA, would you expect to be present in the final miniprep sample?  How could you find out?