Restriction Analysis of pGREEN plasmid from plasmid miniprep

 

Credits:  DNA Learning Center, David Micklos et al.

 

It is standard procedure to identify a plasmid by using DNA restriction analysis.  This involves recovering the plasmid from an overnight culture of E.coli/pGREEN grown in LB/amp broth by using a plasmid miniprep procedure.  Then, a sample of the isolated plasmid from the miniprep experiment along with a control sample of pGREEN are cut with restriction enzymes BamH1 and HindIII.  The two samples are subjected to electrophoresis on the same gel and the bands are compared.  The miniprep DNA should have the same “restriction fingerprint” as the control pGREEN DNA. 

 

Reagents                                                        Equipment

Miniprep DNA/TE                                           micropipettors

0.1  mg/ml pGREEN plasmid                              1.5 ml tubes

BamH1/HindIII enzyme mix                              electrophoresis boxes

5X NEB buffer 2/RNase                                  power supplies

1        X TBE buffer                                              37o C. water bath

Agarose

Distilled water

 

I.                    Restriction Digest.

1.  Label four 1.5 ml tubes as shown in matrix below.  Use the matrix below to add reagents to each tube, in the order given, left to right.  Use a fresh pipet tip for each reagent.

 

Tube

Miniprep DNA

pGREEN

DNA

Buffer/RNase

BamH1/HindIII

Distld H2O

Mini -

5 ml

0

2 ml

0

3 ml

Mini +

5 ml

0

2 ml

2 ml

1 ml

pGREEN +

0

5 ml

2 ml

2 ml

1 ml

pGREEN -

0

5 ml

2 ml

0

3 ml

 

  1. After all reagents are added, close tubes and pool all reagents to bottom of tube by pulsing in microcentrifuge or tapping tube sharply on benchtop.
  2. Place all tubes in 37oC water bath for 30-40 minutes (do not overincubate).

STOP point – you can freeze reaction tubes at -20o C following incubation and thaw when ready to continue.

 

II.                 Gel Analysis of Restriction digest.

  1. Prepare a 0.8% agarose gel to a depth of about 5 mm in casting tray.  Cool till gel sets.  Place in electrophoresis box and cover with TBE buffer and remove comb. 
  2. Obtain reaction tubes from part I (thaw if necessary).  Add 1 ml of loading dye to each tube.
  3. Load 10 ml from each reaction tube into separate well in the gel.  Use a fresh tip to load each sample.  Put in your notebook a diagram that shows which wells contained which sample.
  4. Close box, connect leads to power supply, and electrophorese at 120 volts for 20-40 minutes.  Run is long enough when bromophenol band has moved 30-40 mm from sample wells.
  5. Turn off power supply, remove gel and transfer gel to container for staining.
  6. Stain and view gel.
  7. Analyze results based on the map of the plasmid shown below.

 

 

MAP OF PLASMID pGREEN                                

EcoR1

                                                         0/4528

                                                           |

 

 

 

 

 

 

 


ampr

 

                                                                                                                _BamH1

                                                                                                                   1120                                                                                                        

 

 

                                                                                                            GFP

 

                                                                                                \

                                                                                                 HindIII

                                                                                                   1893

 

 

 

Results and Discussion:

 

Interpreting these gels is not always straightforward.  The miniprep DNA contains impurities so will tend to yield a more “smeared” banding throughout the miniprep lanes.  The smear contains pieces of bacterial DNA as well as RNA and plasmid DNA.  If you see a smear of nucleic acid at the tip of the gel, it may be due to mRNA though the RNase treatment is meant to eliminate most of the RNA. 

It is also confusing to see in the lanes of uncut plasmid DNA as many as three bands when you expect only one.  This occurs because plasmid DNA can exist in three forms:  supercoiled plasmid DNA (tight circle), relaxed plasmid DNA (floppy circle) and linear plasmid DNA.  All these forms have the same molecular weight of 4528 bps, BUT they migrate at different rates through the gel matrix:  the supercoiled DNA runs fastest (farthest from well); the floppy circle runs slowest (closest to well); the linear form moves faster than floppy relaxed circular form but slower than supercoil – hence, in between the two circular forms. 

The first thing to look for is evidence of cutting with restriction enzymes:  look at the mini+ and pGREEN+ lanes that contained restriction enzymes, and verify that you have two bands that run faster than the fastest (most forward) uncut plasmid form.  Uncut plasmid is 4528 bps; cut plasmid should produce a 773 bp piece and a 3755 piece, both of which should run forward to the uncut plasmid. 

Your results:

 

 

 

 

 

Now look at the uncut pGREEN plasmid and the uncut miniprep DNA – how many forms of plasmid DNA can you detect in the control lane (pGREEN-):

 

Describe results in mini- lane:

 

 

Conclusions:

 

 

 

 

Questions:

 

1.      Based on the map of pGREEN above, when you cut the plasmid with BamH1 and HIndIII, you expect to see _______________ bands (number of bands).

2.      The size of the uncut plasmid is __________bps.

3.      The plasmid, when cut with BamH1 plus HindIII, should produce bands of what sizes:_________________________________________

4.  If you cut the plasmid with BamH1 and HindIII and EcoR1, you should see bands of sizes:_______________________________.