Reflection: Classroom Setup Producing a Recombinant Plasmid, pARA-R - Section 1: Introduction



The volume of reagents used in this lab series are nominal and at times the transfer of liquids is difficult to discern with the naked eye. A best practice when working with microvolumes is to pulse spin the reagents for 5 seconds or less in a microcentrifuge to pool the contents of each tube to the bottom of the vessel. This will enable the technician to mix the contents before aliquoting as well as make the contents more visible when transferring.

The water bath incubation step in this lab is critical to the success of the investigation. I would suggest setting up the water bath a few days before the lab, setting to the desired temperature and confirming the temperature with a thermometer and finally calibrating the equipment if needed. It is important that the temperature in the water bath NOT exceed 70C, the optimal temperature for restriction enzymes, as this may lead to the denaturation of the enzyme and thus no restriction or "cleaving" and no ligation or "binding"

  Nobel Prize Lab Teacher's Notes - Volume 2
  Classroom Setup: Nobel Prize Lab Teacher's Notes - Volume 2
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Producing a Recombinant Plasmid, pARA-R

Unit 8: Nobel Prize-Winning Biotechnology
Lesson 2 of 9

Objective: Students will be able to create new recombinant plasmids using DNA Ligase.

Big Idea: If restriction enzymes are the "molecular scissors" of biotechnology then DNA Ligase is the "molecular glue".

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