Reflection: Classroom Setup Confirmation of Restriction and Ligation: Using Agarose-Gel Electrophoresis - Section 3: Methods



Carefully review the reaction matrix for this lab. Observe that you and your students can add water to all five tubes, then add the loading dye to all of the tubes without changing the tip. Then, dispense the plasmid sample into each tube, changing the tip each time to avoid contamination. This is both a time-saving and cost effective approach when completing a large amount of pipetting!

Centrifuge ALL samples to pool the reagents to the bottom of each tube BEFORE aliquoting or transferring liquids using a microcentrifuge. Be certain that the tubes are placed in a balanced configuration if using a table top centrifuge to spin many groups tubes at one time.

The preparation of agarose gels for an entire class can be an enormous task! I suggest preparing the gels several days before the lab and placing the gels with tray and comb in ziploc bag with a little buffer and storing them in the refrigerator (4C) for no more than two days. The most difficult and time consuming part of the lab is the preparation of the gels so reducing time spent and being prepared on lab day is a must!

Finally, when running gels be certain the gel wells are oriented closest to the negative (black) electrode. Additionally be sure to pour enough buffer over the gel until there are no visible dry patches breaking the surface of the buffer especially over the wells. It’s important that the gel be completely under the buffer. However, you don’t want so much buffer in the box that the electrical current cannot run through the buffer or the gel.

  Classroom Setup: Nobel Prize Lab Teacher's Notes - Volume 3
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Confirmation of Restriction and Ligation: Using Agarose-Gel Electrophoresis

Unit 8: Nobel Prize-Winning Biotechnology
Lesson 3 of 9

Objective: Students will be able to use a special staining technique which enables them to visualize DNA fragments in an agarose gel medium.

Big Idea: DNA visualization techniques such as gel electrophoresis is the method of choice when confirming restriction and ligation of DNA.

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Science, Biotechnology, DNA Technology
  145 minutes
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