Producing a Recombinant Plasmid, pARA-R

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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".

Introduction

20 minutes

All of the lab investigations in this unit are based on nothing less than authentic Nobel Prize–winning science. Kary Mullis received the Nobel Prize for his discovery of the Polymerase Chain Reaction or PCR.  Werner Arbor, Daniel Nathans and Hamilton Smith received the Nobel Prize for their work with restriction enzymes. Stanley Cohen, Paul Berg and Herb Boyer received the highly esteemed prize for making the first recombinant DNA molecule. Actually, the recombinant DNA molecule that students create during this lab series extends beyond the scope of these honored Nobel Laureates original work as it incorporates a gene from a eukaryotic rather than prokaryotic organism into a plasmid.  It would be impossible to explain the effect these advancements and breakthroughs have had on the development of biotechnology, medicine, forensic science, and research in general however perhaps by experiencing the same triumphs as these giants the significance will become apparent. 

In this laboratory investigation, the restriction fragments produced during the previous lab, pARA-R Restriction Digest: An Intro to Plasmids and RE's, will be ligated, or bonded together, using DNA ligase, making new recombinant plasmids. The newly formed plasmids will represent recombinant DNA molecules because the four restriction fragments have been recombined in different ways to produce new configurations.

The purpose of this lab is to examine the role of other enzymes critical to the creation of recombinant plasmids, such as DNA Ligase, when performing genetic engineering. 

I begin this lab by setting the context of the work we will be doing and providing background information as illustrated on SLIDES 2 thru 6 of the AMGEN Recombinant DNA Lab Series PowerPoint Presentation. At the end of this segment of our prelab discussion I check for understanding using a Pre Lab Quiz as well as visit the community lab area in our laboratory classroom in order to walk students through staging their workspaces or "bench" as shown in this photo. As we move about the community lab area and either stage or review staging that has already been completed, I explain the significance of each piece of equipment and how it will be used in our work. Students record notes from our discussion in their laboratory notebook as oftentimes the information provided appears on our Post Lab Quiz and Conclusion Questions.

Materials

10 minutes

REAGENTS

Digested pARA (A+ from Lab 2 Restriction Digest)

Digested pKAN-R (K+ from Lab 2 Restriction Digest)

5x Ligation buffer with ATP

T4 DNA Ligase in "lig" tube

Distilled water

 

EQUIPMENT & SUPPLIES

P-20 micropipette and tips

70C water bath

Plastic microfuge tube rack

Permanent marker

Methods

90 minutes

Ligation of pARA/pKAN-R Restriction Fragments

1. Obtain the A+ and K+ tubes from the rack at the front of the class. Place the two tubes in the 70C water bath for 30 minutes. This heat exposure will denature (inactivate) any BamHI and HindIII that might be active. Why is this important?

2. While the tubes are in the water bath, obtain the 5x buffer and a Ligase tube from the instructor. The ligase tube contains 2uL of DNA ligase.

3. After the 30-minute, 70C-incubation step, add 4uL of A+ directly into the DNA ligase at the bottom of the Ligase tube.

4. Using a new tip, add 4uL of K+ tot he solution in the Ligase tube.

5. Using a new tip, and 3uL of 5X ligation buffer directly into the solution at the bottom of the Ligase tube. Discard the buffer tube.

6. Add 2μL of dH2O to the Ligase tube, using a clean tip. Gently and slowly pump the plunger in and out to mix the reagents. Do this without splashing the solution onto the sides of the microfuge tube. This reaction matrix summarizes the contents of the Ligase tube.

7. Briefly pulse spin the contents of the tube to pool the reagents.

8. Place the A+ and K+ tubes in the microfuge racks and keep overnight at room temperature.

Results

30 minutes

This portion of our lab is pretty light as we will need to wait for the confirmation of both the restriction digest and the ligation of the plasmid in the next lab in the series, Confirmation of Restriction and Ligation: Using Agarose-Gel Electrophoresis. We will conduct this lab in order to visualize the results from this lab and assess our technique! Instructors can spend time reviewing the two original plasmid maps and having students make predictions about what will be seen when the DNA samples A+, A-, K+, K-, and Ligase are run on an agarose gel (See sample result here.)

Conclusion

30 minutes

To end this lab investigation students individually answer the Lab 3 Conclusion Questions in preparation for a whole group discussion on the topics presented in this experiment.