LIVING ENVIRONMENT LAB # 2
Aim: How do I perform gel electrophoresis?
per group of 2 students
S 1 agarose gel in casting tray (see previous lab)
S 1 razorblade
S 5 restricted DNA samples (see previous lab)
S 1 DNA size standard (included in biorad kit, HindIII lambda digest)
S DNA sample loading buffer
S 1 2-20μl adjustable micropipet
S 20 pipette tips
S 300 ml 1X TAE electrophoresis-buffer
S 1 electrophoresis gel box
S Blue nontoxic DNA-stain (included in biorad-kit)
S 1 Gel staining tray
S sterile water
S Latex gloves
S 1 300ml graduated cylinder
per group of 4 students
£ 1 power supply
£ 2 black electrical leads
£ 2 red electrical leads
a. Put on latex gloves and wear them throughout the whole procedure.
b. Obtain your prepared agarose gel and your restricted samples.
c. Cut an edge off the upper left hand corner of your gel to orient it.
d. Using a new pipette tip for each sample add 5μl of sample loading dye “LD” to each tube, but the size standard.
e. Close the caps on all the tubes. Mix the components by gently flicking the tubes with your finger.
f. Place the casting tray with the solidified gel in it onto the platform in the gel box. The comb should be at the – end of the box, where the black lead is connected.
g. Pour ca. 275ml of electrophoresis buffer into the electrophoresis chamber. It should just cover the gel.
h. Very carefully, remove the comb from the gel by pulling it straight up. The holes that remain in the gel from the comb are called wells.
i. Gels are read from left to right. The first sample is loaded in the well at the left hand corner of the gel.
j. Using a separate pipette for each sample, load your gel as follows
Lane 1: DNA size marker, clear tube 10μl
Lane 2: Crime Scene, green tube 20μl
Lane 3: Suspect 1, blue tube 20μl
Lane 4: Suspect 2, orange tube 20μl
Lane 5: Suspect 3, violet tube 20μl
Lane 6: Suspect 4, red tube 20μl
Lane 7: Suspect 5, yellow tube 20μl
k. Secure the lid on the gel box. Connect electrical leads to the power supply.
l. Turn on the power supply. St it for 100V and electrophorese the samples for 30 minutes.
m. While you are waiting for the gel to run, you may start with the review questions.
n. When the electrophoresis is complete, turn off the power and remove the lid from the gel box. Carefully remove the gel tray and the gel from the gel box. Nudge the gel off the tray with your thumb and carefully slide it into your plastic staining tray.
o. Pour 60ml of Bio-Safe DNA stain into your plastic staining tray, cover with plastic wrap and let the gel stain overnight.
1. The electrophoresis apparatus creates an electrical field with positive and negative poles at the ends of the gel. DNA molecules are negatively charged. To which electrode pole of the electrophoresis field (+ or -) would you expect DNA to migrate? Explain.
2. What color represents the negative pole?
3. After DNA samples are loaded into the sample wells, they are forced to move through the gel matrix. What size fragments (large vs. small) would you expect to move toward the opposite end of the gel most quickly? Explain.
4. Which fragments (large vs. small) are expected to travel the shortest distance from the well? Explain.
Return to Lesson Plan