Investigative Lab – The Case of the Virulent Virus
Week Two: Is the nucleic acid single- or double-stranded?
The following equipment and reagents will be available, and might be helpful when
designing your experiments. Folders that describe each item in more detail (including
protocols) are available at the front of the room.
S1 Nuclease (degrades single-stranded DNA and RNA)
Thermal cyclers set to 30°C
Agarose gel electrophoresis stuff
Gel boxes
Micropipetters
Micropipette tips
agarose
50X TAE
Pipets and tips
Sterile, deionized water
Gloves
Technique: Cleavage of single stranded nucleic acids with S1 nuclease.
Background
Nucleases are enzymes that digest DNA and RNA into smaller subunits by
disrupting the phosphodiester bonds that hold adjacent nucleotides together.
Exonucleases digest nucleic acids from the ends, while endonucleases cleave internal
phosphodiester bonds.
S1 Nuclease degrades single-stranded DNA and RNA endonucleolytically to yield 5’phosphoryl terminated products. Double-stranded DNA, double-stranded RNA and
DNA-RNA hybrids are resistant to degradation except with extremely high
concentrations of enzyme. (Vogt, V.M. (1973) Eur. J. Biochem. 33, 192.)
Procedures
1. Add 1 g nucleic acid, 1 unit of enzyme, 2L enzyme buffer and sterile
deionized water to at total volume of 20L in a sterile microcentrifuge tube.
2. Incubate at 30°C for 30 minutes
3. Run the reactions on an agarose gel to see if the nucleic acid has been cut by
the enzyme.
Technique: Agarose Gel Electrophoresis
Background
Agarose gel electrophoresis can be used to separate nucleic acids of different
lengths. In general, since nucleic acids are negatively charged, they will migrate towards
a positive electrode in a charged field. Solid agarose forms a porous matrix through
which DNA can migrate at different rates, depending on size. Large molecules will
migrate slowly through the matrix, while small molecules will migrate more quickly.
The size of a nucleic acid can be estimated by comparing its migration to that of known
size standards.
Procedure
To pour a 1% Agarose Gel
1. In a 125 mL erlenmeyer flask add:
0.5g agarose
50 mL 1xTAE
2. Microwave for 2 min or until agarose is in solution. Swirl the flask gently,
every 15 sec., to prevent boiling over in the microwave. The gel solution may
superheat, so take care when swirling!
3. Add: 5µL EtBr solution (5.0 mg/mL)
EtBr is a known mutagen! Wear gloves, and report spills immediately!
4. Pour molten gel into gel mold with an appropriate comb in place. Let set for at
least 30 minutes. Small air bubbles can be removed before gel starts to set using a
small pasteur pipet.
5. Fill gel buffer tank with 280 mL 1XTAE and remove gel comb
6. Load up to 20L of each nucleic acid sample, in 1X sample buffer, into a well
as illustrated below.
LOADING AGAROSE GELLS
7. Carefully slide the top on the gel box.
8. Plug the red lead into the red (+) terminal and the black lead into the
black (-) terminal.
9. Turn the power supply on to 100 volts.
10. Run the gel until the blue dye has migrated down 2/3 of the length of the gel.
(up to 1 hour)
11. When the gel has run, turn off the power supply. NEVER
OPEN THE GEL BOX IF THE POWER IS STILL ON.
12. Remove the gel cassette (with gel) from the electrophoresis
chamber. Make sure the gel is only handled with gloved hands.
13. Place the gel on the UV Transilluminator. WARNING:
PROTECTIVE EYE WEAR MUST BE WORN AT THIS TIME!!! UV
LIGHT WILL SERIOUSLY BURN UNPROTECTED EYES!!!
14. Take a picture of the gel by placing the camera apparatus over the gel,
opening up Kodak’s ID program, and performing any other manipulations you
deem necessary.
15. Save a picture of the gel on your disc, and make sure it is part of the final
report!