laboratory 4: making sure you have created a recombinant plasmid lssi alum, lindsey engle, granite...

LABORATORY 4: MAKING SURE YOU HAVE CREATED A RECOMBINANT PLASMIDLSSI Alum, Lindsey Engle,Granite Hills High SchoolEl Cajon, CA

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What have we already done?

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

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Restriction analysis of pKAN-R and pARA

BamHIpKAN-R5,512 bp

pARA4,872 bp

PBAD-rfp 806 bp

376 bp

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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

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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.

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Gel Electrophoresis for DNA

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In your lab notebook write down the answers to these pre-lab questions, and discuss your answers with your partner.

1. Why do DNA restriction fragments and plasmids separate when analyzed by gel electrophoresis?

2. Why is it important to identify and verify a recombinant plasmid?

3. When DNA fragments are joined with a DNA ligase an array of products is created. How does this happen?

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

• Vocabulary Activity

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Chapter 4: Making Sure You’ve Got What You Need

• Learning Goals– Describe the importance of verifying products

created in the genetic engineering process– Predict the relative speed of DNA fragments

and plasmids in gel electrophoresis– Separate and identify DNA restriction

fragments and plasmids using gel electrophoresis

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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

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Lab Prep & Aliquoting GuidelinesReagents/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

12ul/group RT Protect from light keep in amber tubesInvert or finger vortex tube to ensure solution is mixed before aliquoting

6 L of 1x SB buffer/kit RT Please return bottles10 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/Supplies10 Student boxes with the following:

1 p20 micropipette 1 microfuge rack1 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 centrifuges10 Gel Electrophoresis chambers 10 Gel trays 10 Gel combs5 Power supplies1 Embi Tec/PrepOne System

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SafetyGeneral 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.

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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

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• Prepare samples (see Table 4.1)• Load and run gels (40-50 min.)

– Students load samples in same order

• Remove gels and photograph (teacher)

Student Workflow

Table 4 .1: Addition of reagents to the geK- , geK+, geA- , geA+, and geLJG tubes

SequencegeK-tube

geK+tube

geA-tu be

geA+

geLIGtube

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 N ond i ges ted pAR A (A- )

Step 7 Digested pAR A (A+)

4 µL

4 µL

S tep 8 Ligated p l a s m i d (L IG) 5 µL

4 µL

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Running Agarose Gel

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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.

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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.

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Making a 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. . .

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Lab 4 Results

• Draw your gel electrophoresis results in the same way as your hypothesis.

Hint: Your students can take pictures of the results, print and paste in their lab reports.

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• Compare your hypothesis and results.• Was the digestion successful?

– What is your evidence?• Was the ligation successful?

– What is your evidence?

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Restriction analysis of pKAN-R and pARA

Restriction fragments after digest with Hind III and BamH I

4,706 bp

BamH I Hind III

806 bp

BamH IHind III

376 bp

BamH IHind III

BamH I Hind III

4,496 bp

M K- K+ A- A+ Lig DyeResults:

(Smaller fragment not observed)

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Analysis

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

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Plasmid Size and Shape Will Influence Migration

Through Gel• Many possible constructs from ligation

– Any complimentary sticky ends may recombine

• Plasmids shapes may differ:

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• 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 band in the A+ column (at 4495 bp) shows the plasmids were digested. The band of the digested pARA at 377 bp was not observed but the presence of the larger fragment shows the plasmid was digested.

• Presence of pARA-R confirms that the ligation has been successful.

Lab 4 Conclusions

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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