laboratory 4: making sure you’ve created a recombinant plasmid LSSI Alum, Lindsey Engle LSSI Alum, Shawn Hurley LSSI Alum, Mary Haus

What have we done up to this point? In your own words, in your lab notebook, briefly write down what we accomplished in Lab 2. (You have 2 minutes) We used restriction enzymes (BamHI and HindIII) to “digest” or cut the pKAN-R plasmid and pARA plasmid. Why did we do this? To remove the rFP gene and its promoter To open the pARA plasmid so we can insert the rFP gene

Restriction analysis of pKAN-R and pARA 5,512 bp BamHI pARA 4,872 bp PBAD-rfp 806 bp 376 bp

Let’s Reflect Some More In your own words, in your lab notebook, briefly write down what we accomplished in lab 3. (2 minutes) We used DNA ligase to form a recombinant plasmid called pARA-R. **If we were successful! Why did we do this? So that we can give this plasmid to E. coli to express the red fluorescent protein gene through transformation!

Recombinant construct of interest

How will we know if we were successful? 1. We must confirm the digestion of the pKAN-R and pARA plasmids using a control for comparison. 2. We must confirm the ligation has successfully created the pARA-R plasmid.

Background – Gel Electrophoresis https://www.dnalc.org/resources/animations/gelelectrophoresis.html http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.isolatedna/electrophoresis-and-gel-analysis/

In your lab notebook write down the answers to these pre-lab questions, and discuss your answers with your partner. Why do DNA restriction fragments and plasmids separate when analyzed by gel electrophoresis? Why is it important to identify and verify a recombinant plasmid? When DNA fragments are joined with a DNA ligase an array of products is created. How does this happen?

Before we begin, let’s review some terms. . . Vocabulary Activity

Complete Genetic Engineering Sequence Lab 1: Tools of the Trade – Pipetting and Gel Electrophoresis Lab 2: Restriction Digest (cut pKAN-R and pARA) Lab 3: Ligation (combine pieces to make pARA-R) Lab 4: Confirmation (gel electrophoresis) Lab 5: Transformation (introduce pARA-R into bacterial host cells) Lab 6: Culture transformed bacteria, isolate and purify the protein

Goals of Lab 4 Logistical (students will coordinate procedural steps necessary to): Confirm the presence/identity of pARA-R plasmid by separating DNA restriction fragments and plasmids using agarose gel electrophoresis Educational (students will be able to): Describe the importance of verifying products generated in the engineering process Predict the relative migration speed of DNA restriction fragments & plasmids through a gel during electrophoresis based on their sizes

Chapter 4: Suggested Activity Sequence Session 1: Active reading and class discussion pp 69-74 Session 2: Lab 4 “Verification of Restriction and Digestion Using Gel Electrophoresis” pp 75-79 Session 3: Analyze gel pictures; Ch. 4 questions p 80

Lab Prep & Aliquoting Guidelines Reagents/Supplies Aliquot Storage Temp Notes 140 ul LD/class Loading Dye (LD) The (LD) contains the gel green stain - please see note before using 12ul/group RT Protect from light keep in amber tubes until ready to use. Invert or finger vortex tube to ensure solution is mixed before aliquoting 130 ul M/class (M) The Marker (M) is ready to use – please see note before using Protect from light keep in amber tubes 6 L of 1x SB buffer/kit   Please return bottles 10 gels per class. If you prefer to have your students pour their own gels, please let us know when you make your reservation on LARS. N/A 4o Please return any unused gels. Discard used gels in the trash. Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 waste and 1 ice bucket 1 box of refillable tips (2 ul-200 ul) 4 Mini centrifuges 10 Gel Electrophoresis chambers 10 Gel trays 10 Gel combs 5 Power supplies 1 Embi Tec/PrepOne System

Safety General Lab Safety Guidelines Use laboratory coats, safety glasses and gloves as appropriate. Avoid restrictive clothing and open-toed shoes. No eating or drinking in the lab. Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment. Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab. Wash hands at the conclusion of the lab. Lab Safety Guidelines for lab 4 Follow specific safety and disposal guidelines for the DNA gel green DNA stain or the DNA stain you are using. Use caution with hot liquids and glassware. Wear heat-proof gloves to move hot glassware and liquids. Make sure the power is turned off on power supplies before connecting electrodes. After gel run is complete, turn off power supply then unplug electrodes. If the power is on, do not touch the buffer or electrophoresis equipment as you may receive an electrical shock. Never leave the electrophoresis power unit on without supervision. There is a risk of fire if the buffer leaks out or if the buffer should evaporate completely during electrophoresis.

Lab 4: Verification of Restriction and Digestion Using Gel Electrophoresis Method Overview: Use gel electrophoresis to separate and visualize DNA fragments from Lab 2 and recombinant plasmids from Lab 3 Analyze sizes of fragments and plasmids using DNA ladder (marker) and control lanes

Student Workflow Prepare samples (see Table 4.1) Load and run gels (40-50 min.) – Students load samples in same order Remove gels and photograph (teacher)

Table 4.1:Addition of reagents to the geK-, geK+, geA-, geA+, and geLIG tubes Sequence tube Step 4 and 5 Distilled water (dH2O) 4 µL 4 µL 4 µL 4 µL 3 µL S tep6 Loading dye (LD) - - Step 6 Nondigested pKAN -R (K-} 2 µL 2 µl 2 µL 2 µL 2 µL 4 µL S tep 7 Digested pKAN-R (K+) S tep 7 Nondigested pARA (A-) Step 7 Digested pAR A (A+) 4 µL 4 µL 4 µL S tep 8 Ligated plasmid (L IG) 5 µL

Making an Agarose Gel http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.agarosemake/making-an-agarose-gel/

Loading & Running an Agarose Gel http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.agaroserun/running-an-agarose-gel/

Virtual AGE Lab Optional activity to give students an opportunity to become more familiar with the steps of making and running an agarose gel in a simulated environment http://learn.genetics.utah.edu/content/labs/gel/

Making an Hypothesis Based on your knowledge of labs 2 and 3, draw your gel, label each lane with what was added, and predict the resulting bands that will be seen if we are successful with the digestion and ligation. Refer to the background information in the student guide. Label your diagram “Hypothesis.” Here is a sample diagram. . .

Follow the lab protocol/methods in the student guide The Loading Dye (LD) contains the DNA stain-Gel Green. Students should follow the procedure in the table on page 77 to prepare samples for loading into gel wells. The DNA Marker/Ladder (M) has been prepared and is ready to use.

Photographing Gels When gel run is complete (approximately 15-20 minutes), a good way to determine if the gels have run long enough is to observe the orange G (OG) which will be visible on the gel while it is running. Once the orange G is ~ 1/3 of the way down the gel, the gels are ready to be photographed. Plan system to track gels by period and group number Use the PrepOne system to visualize and photograph the gels. Allow students to use cell phones to capture images of the gels or teacher can capture images using his/her phone.

Lab 4 Results Draw your gel electrophoresis results in the same way as your hypothesis. Tip: Your students can take pictures of the results, print and paste in their lab reports.

Analysis Compare your hypothesis and results. Was the digestion successful? What is your evidence? Was the ligation successful?

Restriction analysis of pKAN-R and pARA Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4,706 bp BamH I Hind III 4,496 bp Hind III BamH I 806 bp Hind III BamH I 376 bp

Results: (Smaller fragment not observed)

Going Further What are some possible explanations for your results if they were different than you predicted?

Plasmid Size and Shape Will InfluenceMigration Through Gel Many possible constructs from ligation – Any complimentary sticky ends may recombine Plasmids shapes may differ:

Lab 4 Conclusions The original plasmids without the enzyme are present. The multiple bands in the K- and A- columns result from various conformations of the plasmid. The plasmid (due to handling and replication) may have several possible forms such as supercoiled, open-circle or multimer. The digest was successful. The bands in K+ column (at 4705 bp and 807 bp) and the bands in the A+ column (at 4495 bp and 377 bp) indicate the plasmids were digested. Presence of pARA-R confirms that the ligation has been successful.

DNA Separation Lab with Betty Burkhard Shows what it’s like to do these labs in a classroom setting Talks about the importance of gel electrophoresis training for students