pARA-R Restriction Digest: An Introduction to Plasmids and Restriction Enzymes

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Objective

Students will be able to examine the role of restriction enzymes (RE's) and their importance in genetic engineering as well as demonstrate their understanding by using the RE's BamHI and HindIII to isolate a gene!

Big Idea

Discover the two most powerful yet fundamental tools used in biotechnology!

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 enzymes BamHI and HindIII are used to digest the recombinant plasmid, pARA-R. The restriction digest will isolate from pARA the rfp gene from the larger fragment of the plasmid that containing ampr, araC, and pBAD. The protocol uses a control, undigested pARA-R, along with a DNA size marker or ladder that will help identify and confirm the sizes of the restriction fragments.

The purpose of this lab is to examine the role of restriction enzymes and their importance in genetic engineering as well as examine the use of bacterial plasmids and their role in 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

5 minutes

REAGENT

pARA (80 ng/uL)

pKAN-R (80 ng/uL)

Restriction enzymes (BamHI + Hind III)

2.5x restriction buffer

Distilled water, dH2O

 

EQUIPMENT & SUPPLIES

P-20 micropipette and tips

1.5mL microfuge tubes

minicentrifuge

37C water bath

Permanent marker

Methods

60 minutes

Preparing the pARA-R restriction digest

1. Obtain the following four microfuge tubes: pARA, pKAN-R, BamHI and HindIII (enzyme mix) and 2.5x buffer.

2. Obtain four clean 1.5mL microfuge tubes and use a marker to label a set of four 1.5 tubes as follows:

A + = pARA + BamH I and Hind III

A - = undigested pARA (pARA without enzyme)

K+ = pKAN-R+ BamH I and Hind III

K- = undigested pKAN-R(pKAN-R without enzyme)

3. This reaction matrix summarizes the reagents used in the restriction digest. To set up the digest, follow the specific directions beginning at Step 4 below.

4. Use a fresh tip and add 4uL of 2.5x restriction buffer to all four tubes.

5. Add 2uL of dH2O to tubes labeled A- and K- (What is the purpose of these two samples?)

6. Use a fresh tip and add 4uL of pARA to tubes labeled A+ and A-.

7. Use a fresh tip and add 4uL of pKAN-R to tubes labeled K+ and K-.

8. Add 2uL of enzyme mix, containing BamHI and HindIII, to the A+ and K+ tubes.

9. If a minicentrifuge is available, set the tubes into the rotor, being certain the tubes are in a balanced configuration, and pulse spin the tubes for four seconds. This brief spin will pool all of the reagents to the bottom of each tube.

10. Place all four tubes into the 37C water bath and incubate for at least 60 minutes.

11. Following the 60 minute incubation, the digest can be kept frozen, at -20C, until confirmation of the ligation is performed via gel electrophoresis.

Results

30 minutes

This portion of our lab is pretty light as we will need to wait for the confirmation of the ligation of the plasmid in a future lab in order to analyze the results and assess our technique! However instructors can spend time reviewing the two original plasmid maps and having students make predictions about the construction of the new plasmid. 

Conclusion

30 minutes

To end this lab investigation students individually answer the Lab 2 Conclusion Questions in preparation for a whole group discussion on the topics presented in this experiment.