Gel Electrophoresis Lab
Purpose- To determine DNA fragment length of DNA cut with various enzymes.
Background- What is DNA? What are restriction enzymes? Why do scientist use gel
electrophoresis? How does gel electrophoresis work? How do you determine fragment
lengths using gel electrophoresis?
Teacher prep- melt agarose in hot water bath
Materials- micropipette, ladder lambda DNA, lambda DNA cut with various enzymes,
uncut lambda DNA, gel chamber and mold, comb, electricity source, buffer, agarose,
micro-centrifuge, Carolina BLU stain
Procedure1. Prepare mold, by placing on end pieces and comb.
2. Pour melted agarose gel into mold, and allow time to solidify. (May take 20-30
minutes, so move on to step 3).
3. Buffer and loading dye have already been added to the DNA samples. Use the microcentrifuge to bring contents of DNA tubes to the bottom.
4. Once gel is set, carefully remove ends. Leave the comb in. Set gel tray in chamber
and add buffer solution. Buffer should be poured until in just reaches over the gel. Very
gently ease the comb from the gel. Do not tear the wells.
5. Draw up 20 uL of the lambda DNA ladder using the micropipette, and insert into the
1st well.
6. Get a new tip. Draw up 20uL of the lambda DNA cut with HindIII and insert into the
2nd well.
7. Get a new tip. Draw up 20uL of the lambda DNA cut with EcoRI and insert into the
3rd well.
8. Get a new tip. Draw up 20uL of the uncut lambda DNA and insert into the 4th well.
9. Hook up power source. Make sure the positive charge is connected farthest from the
wells.
10. Leave the gel to run for a couple of hours. Watch during the process. Do not let the
DNA run off the gel.
11. Pour off the buffer solution. Pour about 10mL of Carolina BLU onto the surface of
the gel. Leave for 4 minutes.
12. Use cold water to rinse gel.
Analysis Questions:
Use the lambda DNA ladder to compare the unknown sequences. How many times does
HindIII cut the DNA? How many times does EcoRI cut the DNA? Do the cuts aligned
with the fact that smaller pieces of DNA move further down the gel. Add up the
fragment lengths. Do they total to be the same amount of base pairs in length?