The Biotechnology Education Company - EDVOTEK · The Biotechnology Education Company ... • Mortar...

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

The Biotechnology Education Company ®

907.060419

EDVO-Kit #

907Isolation and

Gel Analysis ofDNA from Plants

Storage: See Page 3 for specifi c storage instructions

EXPERIMENT OBJECTIVE:

The experimental objective is to isolate genomic DNA from plant tissue.

SAMPLE LITERATURE

Please

refer

to in

cluded

weblin

k for c

orrect

versi

on.

Isolation and Gel Analysis of DNA From Plants

907.060419

2

xxx907EDVO-Kit #

EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Table of Contents

Page

Experiment Components 3

Experiment Requirements 3

Background Information 5

Experiment Procedures

Experiment Overview and General Instructions 7

Isolation of Plant DNA 8

Electrophoresis

Agarose Gel Preparation 10

Conducting Electrophoresis 14

Staining and Visualization of DNA 17

Method 1: Staining with InstaStain® Methylene

Blue Cards

Method 2: One-Step Staining and Destaining

with InstaStain® Methylene Blue

Method 3: Liquid Staining with Methylene Blue Plus™

Study Questions 21

Instructor's Guidelines 23

Notes to the Instructor 24

Pre-Lab Preparations

DNA Isolation 26

Electrophoresis 28

Quantity Prep for Agarose Gel Electrophoresis 29

Experiment Results and Analysis 30

Study Questions and Answers 31

Safety Data Sheets can be found on our website:

www.edvotek.com/safety-data-sheets

All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor adminis-tered to or consumed by humans or animals.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment com-ponents are derived from human sources.

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

This experiment contains reagents and materials for ten groups.


907.060419

3

EDVOTEK • The Biotechnology Education Company® 1.800.EDVOTEK • www.edvotek.com

FAX: 202.370.1501 • email: [email protected]

907EDVO-Kit #

Experiment Components

Contents A. Extraction Buffer 2X B. β-mercaptoethanol C. Ammonium acetate pH 7.5 D. TE buffer (10X) E. Standard Genomic DNA [50 μg/ml]

• Pea Seeds • Practice Gel Loading Solution • 10x Gel Loading Solution • UltraSpec-Agarose™ powder • Concentrated electrophoresis buffer • InstaStain® Methylene Blue • Methylene Blue Plus™ • Practice Gel Loading Solution • 1 ml and 10 ml pipets • Transfer Pipets • Microcentrifuge tubes • Spooling rods

• Horizontal gel electrophoresis apparatus • D.C. power supply • Automatic micropipets with tips • Balance • Visible white light visualization system • Shaker platform or vortex or (optional) • Microcentrifuge • Sorvall Centrifuge (or similar) • Centrifuge tubes and caps • Pipet pump • Pasteur or capillary pipets • Mortar and pestle • Test tubes (13 x 100 mm) • Glassware (fl asks, beakers) • Ice bucket and ice • Horticulture grade vermiculite • Plastic wrap • Distilled water • Isopropanol (70 or 95%) or Ethanol (100%)

Requirements

Components A-E must be stored at

-20°C

This experiment contains reagents and materials for ten groups


907.060419

4

xxx907EDVO-Kit #


5Isolation and Gel Analysis of DNA From Plants

907EDVO-Kit #

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Duplication of any part of this document is permitted for non-profi t educational purposes only. Copyright © 1999-2006 EDVOTEK, Inc., all rights reserved. 907.060419

Bac

kgro

und Info

rma

tion

Background Info Title

The isolation of high molecular weight chromosomal DNA is often the fi rst step in molecular cloning. Large DNA is very sensitive to mechanical shear which causes random breaks in the phosphate bonds of the mol-ecule. If the extraction procedure is performed carefully, large fragments of chromosomal DNA can be obtained with an average fragment length of 100,000 to 200,000 base pairs. Since the average length of a gene is about 2,000 base pairs, there is a high probability that genes of interest will remain intact in fragments of DNA. In subsequent steps, specifi c genes can be cloned.

Biologists recognize that cells are the basic units of structure for all living things. The cells of prokaryotes (bacteria) are relatively homogeneous. Their cytoplasm is differentiated into regions that differ in appearance, but these regions are not physically distinct and are not subdivided by physical barriers like membranes. Eukaryotic cells have several smaller, membrane-bound structures. These include the nucleus, mitochondrion, and chloroplast called organelles. Organelles are physically distinct struc-tures within the cytoplasm of the cell but separated from it.

Both mitochondria and chloroplasts contain their own DNA which carries the information necessary for these organelles to produce enzymes to perform their functions. This is a small proportion of the total DNA found in a plant cell. The bulk of DNA is located in the cell nucleus.

The objective of this experiment is to isolate plant chromosomal DNA with minimum breaks. This procedure was tested on various specimens of plants and generally good results are obtained with most leaf tissue. Leaves from phenolic-rich plants such as roses, strawberries and rho-dodendrons, as well as leaves from succulent plants such as aloe and cactus are problematic and are not recommended for this extraction procedure.

This procedure works well with peas, sunfl ower, and clover. The resuspend-ed cells are fi rst mixed with a solution of ethylenediamine tetraacetic acid (EDTA) which forms complexes (chelates) with metal ions. Divalent metal cations, such as Mg+2 are the required cofactor for majority of DNases. In the presence of EDTA, DNA being extracted is protected from DNase degradation since the complexed Mg+2 cannot be utilized by the DNases. Detergent dissolves the cell membrane and denatures many proteins. The aqueous cell lysis solution is overlaid with cold isopropanol or ethanol. High molecular weight DNA is spooled onto a glass rod when it is mixed at the interface of the two liquids. Due to its size and abundance, chro-mosomal DNA forms a viscous mass that is easily collected.

6



EDVO-Kit #


Bac

kgro

und

Info

rma

tion

Background Info Title

Agarose gel electrophoresis is a convenient method for analyzing DNA in this size range. The gel consists of microscopic pores that act as a mo-lecular sieve. DNA samples are loaded into wells made in the gel during casting. The gel is placed in an electrophoresis chamber containing a buffer. Direct current is applied from a power supply. Since DNA has a strong negative charge at neutral pH, it will migrate through the gel towards the positive electrode. Pores in the agarose gel separate linear fragments of DNA according to their size. The smaller the fragment, the faster it will migrate. If many fragments are present in a wide range of sizes (such as occurs with badly sheared chromosomal DNA) the DNA ap-pears as a smear instead of a distinct band after electrophoresis.


907EDVO-Kit #



The Exp

erim

ent

EXPERIMENT OBJECTIVE:

The experimental objective is to isolate genomic DNA from plant tissue.

LABORATORY SAFETY

1. Gloves and goggles should be worn routinely as good laboratory practice.

2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the labora-tory.

5. Always wash hands thoroughly with soap and water after handling reagents or bio-logical materials in the laboratory.

LABORATORY NOTEBOOK RECORDINGS:

Address and record the following in your labo-ratory notebook or on a separate worksheet.

Before starting the Experiment:

• Write a hypothesis that refl ects the experiment. • Predict experimental outcomes.

During the Experiment: • Record (draw) your observations, or photograph the results.

Following the Experiment: • Formulate an explanation from the results. • Determine what could be changed in the experiment if the ex-

periment were repeated. • Write a hypothesis that would refl ect this change.

Experiment Overview and General Instructions

8



EDVO-Kit #


The

Exp

erim

ent

Isolation of Plant DNA

ISOLATE PLANT DNA

1. Grind 0.5 grams dried (air-dried or freeze-dried) pea shoots (leaves) to a fi ne powder in a mortar with pestle (use mostly leaf tissue; try to remove stems).

2. Mix the powder with 10 ml extraction buffer in a 50 ml, capped centrifuge tube.

3. Place the tube into a 65°C waterbath and incubate for 1 hour. Several times during the hour, mix the tube's contents by inversion.

This procedure was tested using pea seedlings and various other specimens of plants. Gen-erally, good results are obtained with most leaf tissue, especially peas, sunfl ower and clover. Leaves from phenolic-rich plants such as rose leaves, strawberries and rhododendrons, as well as succulent plants such as aloe and cactus, are not recommended for this extraction procedure. DNA extracted from the plant tissue can be analyzed by electrophoresis on an agarose gel, but should not be expected to cut well by restriction endonucleases.

Place the Isopropyl rubbing alcohol on ice before the lab starts.

Allow adequate time to equilibrate a water bath at 65°C.

Use a fresh pipet when using different stock solutions to avoid cross contamination.

4. Place tubes in a beaker fi lled with cool tap water for 10 minutes.

5. Centrifuge at 6000 rpm for 10 minutes to break the emulsion and separate the tube contents into two phases.

6. Transfer the supernatant to a fresh tube or beaker (you may wish to fi lter the supernatant).

Note: The supernatant may be dark green in this preparation making the precipitation of DNA somewhat more diffi cult to see. If this is a problem, the supernatant can be diluted with additional extraction buffer or TE buf-fer before the precipitation step.

7. To the aqueous phase in the tube or beaker, add 2/3 volume of ice cold isopropyl alcohol. Allow the alcohol to stream down the inside wall of the vessel.

Example: if you have transferred 24 ml to the fl ask, add 2/3 x 24 or 16 ml of isopropyl alcohol.

8. Using a spooling rod, gently stir the mixture at the interface of the isopropanol/aqueous DNA solution. Twirl the rod while stirring to spool out the DNA. Occasionally remove it to see if DNA is being collected.


907EDVO-Kit #



The Exp

erim

ent

Isolation of Plant DNA

9. Rinse the spooled DNA with 95% ETOH to remove proteins and facili-tate resuspension of the DNA.

10. Transfer the DNA to a clean 15 ml tube (scrape the DNA off the spool-ing rod into the tube).

11. Add 4 ml of "DNA Wash Solution" and swirl to wash.

12. After about 20 minutes, transfer the precipitated DNA to a microcen-trifuge tube and centrifuge briefl y to drive the DNA to the bottom of the tube.

13. Using a pasteur or capillary pipet, remove the residual liquid. Allow the DNA to dry in the uncapped tube for about 10 minutes on the bench top.

12. Add 0.75 ml of TE (Tris-EDTA) buffer to the DNA to dissolve the precipi-tate.

13. Large quantities of DNA will require some time to dissolve completely. To expedite the process, place the tubes on a shaker platform or vor-tex (set on low/medium).

Otherwise, refrigerate for several days or longer. If DNA is not com-pletely dissolved, take aliquots of solution and proceed with agarose gel analysis.

Note:

If you do not have a 0-10μl micropipet, prepare a con-centrated DNA solution and make serial dilutions of the DNA. Load the vari-ous dilutions.

PREPARING SAMPLES FOR ELECTROPHORESIS

1. Label 6 microcentrifuge tubes 1, 2, 4, 10, and 20.

2. Transfer 1 μl, 2 μl, 4 μl, 10 μl, and 20 μl of the dissolved DNA to the appropriate tubes.

3. Add TE (Tris-EDTA buffer) to each tube to reach a fi nal volume of 35 μl.

4. Add 5 μl of the 10x gel loading solution to each tube.

10



EDVO-Kit #


The

Exp

erim

ent

AGAROSE GEL REQUIREMENTS FOR THIS EXPERIMENT

• Recommended gel size: 7 x 7 cm or 7 x 14 cm

• Number of sample wells required: 6

• Placement of well-former template: fi rst set of notches

• Agarose gel concentration: 0.8%

PREPARING THE GEL BED

1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape.

A. Using Rubber dams:

• Place a rubber dam on each end of the bed. Make sure the rubber dam fi ts fi rmly in contact with the sides and bottom of the bed.

B. Taping with labeling or masking tape:

• With 3/4 inch wide tape, extend the tape over the sides and bottom edge of the bed.

• Fold the extended edges of the tape back onto the sides and bottom. Press contact points fi rmly to form a good seal.

2. Place a well-former template (comb) in the fi rst set of notches at the end of the bed. Make sure the comb sits fi rmly and evenly across the bed.

Electrophoresis - Agarose Gel Preparation


907EDVO-Kit #



The Exp

erim

ent


CASTING THE AGAROSE GEL(S)

3. Use a 250 ml fl ask or beaker to prepare the gel solution.

Amt ofAgarose

(g)

ConcentratedBuffer (50x)

(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 14

0.23

0.46

0.6

1.2

29.4

58.8

30

60

+ =+

Individual 0.8%* UltraSpec-Agarose™ Gel

DNA Staining with InstaStain® MetBlue

Table

A.1

*0.77% UltraSpec-Agarose™ gel percentage rounded up to 0.8%

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 14

0.23

0.46

30

60

+

Table

A.2 Individual 0.8%* UltraSpec-Agarose™ Gel

DNA Staining with InstaStain® MetBlue


If preparing the gel with concentrated (50x) buffer, use Table A.1.

IMPORTANT

Check with your instructor regarding the concentration of the buffer you are using to prepare your gel. Use the appropriate table (A.1 or A. 2) below.

If preparing the gel with diluted (1x) buffer, use Table A.2.

4. Swirl the mixture to disperse clumps of agarose powder.

5. With a marking pen, indicate the level of the solution volume on the outside of the fl ask.

Diluted buffer is onevolume of concentrated buffer to every 49 volumes of distilled or deionized water. See Table B.

12



EDVO-Kit #


The

Exp

erim

ent

6. Heat the mixture to dissolve the agarose powder. The fi nal solution should appear clear (like water) without any undissolved particles.

A. Microwave method:

• Cover the fl ask with plastic wrap to minimize evaporation.

• Heat the mixture on High for 1 minute.

• Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is completely dissolved.

B. Hot plate method:

• Cover the fl ask with aluminum foil to prevent excess evapora-tion.

• Heat the mixture to boiling over a burner with occasional swirling. Boil until all the agarose is completely dissolved.

Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.


At high altitudes, it is recommended to use a microwave oven to reach boiling temperatures.

7. Cool the agarose solution to 60°C with careful swirling to promote even dissipa-tion of heat. If detectable evapora-tion has occurred, add distilled water to bring the solution up to the original volume marked on the fl ask in step 6.

After the gel is cooled to 60°C:

If you are using rubber dams, go to step 9.

If you are using tape, continue with

step 8.

8. Seal the interface of the gel bed and tape to prevent the agarose solution from leaking.

• Use a transfer pipet to deposit a small amount of cooled agarose to both inside ends of the bed.

• Wait approximately 1 minute for the agarose to solidify.

9. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface.

10. Allow the gel to completely solidify. It will become fi rm and cool to the touch after approximately 20 minutes.

DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED.

Hot agarose solution may irreversibly warp the bed.

60˚C


907EDVO-Kit #



The Exp

erim

ent


PREPARING THE GEL FOR ELECTROPHORESIS

11. After the gel is completely solidifi ed, carefully and slowly remove the rubber dams or tape from the gel bed.

Be especially careful not to damage or tear the gel wells when removing the rubber dams. A thin plastic knife, spatula or pipet tip can be inserted between the gel and the dams to break possible surface tension.

12. Remove the comb by slowly pulling straight up. Do this carefully and evenly to prevent tearing the sample wells.

13. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.

14. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer.

For DNA analysis, the recom-mended electrophoresis buf-fer is Tris-acetate-EDTA, pH 7.8. The formula for diluting EDVOTEK (50x) concen-trated buffer is one volume of buffer concentrate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit.

50x Conc.Buffer (ml)

DistilledWater (ml)

6

8

10

20

294

392

490

980

+EDVOTEKModel #

Total Volume Required (ml)

Electrophoresis (Chamber) Buffer

M6+

M12

M36 (blue)

M36 (clear)

300

400

500

1000

Dilution

Table

B

IMPORTANT: Check with your instructor to determine if the buf-fer has previously been diluted. Pour the appropriate amount of 1x buffer into the electrophoresis chamber according to Table B below.

15. Make sure that the gel is completely submerged under buffer before proceeding to loading the samples and conducting electrophoresis.

14



EDVO-Kit #


The

Exp

erim

ent

Electrophoresis - Conducting Electrophoresis

PRACTICE SAMPLE DELIVERY (GEL LOADING)

Accurate sample delivery technique ensures the best possible gel results. Pipeting mistakes can cause the sample to become diluted with buffer, or

cause damage to the wells with the pipet tip while loading the gel.

If you are unfamiliar with loading samples in agarose gels, it is recom-mended that you practice sample delivery techniques before conducting the actual experiment. EDVOTEK electrophoresis experiments contain practice gel loading solution for this purpose. It is highly recommended that a separate agarose gel be cast for practice sample delivery. One suggested activity is outlined below:

1. Cast a gel with the maximum number of wells possible.

2. After the gel solidifi es, place it under buffer in an electrophoresis ap-paratus chamber.

Alternatively, your teacher may have cut the gel into sections be-tween the rows of wells. Place a gel section with wells into a small, shallow tray and submerge it under buffer or water.

Note: The agarose gel is sometimes called a "submarine gel" because it is submerged under buffer for sample loading and electrophoretic separation.

3. Practice delivering practice gel loading solution to the sample wells. Take care not to damage or puncture the wells with the pipet tip. Follow guidelines below for delivering DNA samples to be stained with InstaStain® Methylene Blue.

If you are using a

• Variable automatic micropipet: Load the sample well with 35-38 microliters of sample.

• Transfer pipet: Load each sample well until it is full.

4. If you need more practice, remove the practice gel loading solution by squirting buffer into the wells with a transfer pipet.

5. Replace the practice gel with a fresh gel for the actual experiment.

Note: If practice gel loading is performed in the electrophoresis chamber, the small amount of practice gel loading solution delivered to the wells will not interfere with the experiment. It is not necessary to prepare fresh buffer.

If you are using transfer pipets, gently squeeze the pipet stem, instead of the bulb to help con-trol the delivery of small sample volumes.


907EDVO-Kit #



The Exp

erim

ent

LOAD THE SAMPLES

1. Obtain a tube of Standard Genomic DNA Marker from your instructor.

2. Load 35 to 40 μl of each DNA sample in the following manner:


Reminder:

During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

+Black Red

Sample wells

–

RUNNING THE GEL

1. After the DNA samples are loaded, carefully snap the cover down onto the elec-trode terminals.

Make sure that the negative and positive color-coded indicators on the cover and apparatus chamber are prop-erly oriented.

2. Insert the plug of the black wire into the black input of the power source (negative input). Insert the plug of the red wire into the red input of the power source (positive input).

Lane Tube 1 1 1 μl plant DNA 2 2 2 μl plant DNA 3 4 4 μl plant DNA 4 10 10 μl plant DNA 5 20 20 μl plant DNA 6 Marker Genomic DNA Marker [50 μg/ml]

16



EDVO-Kit #


The

Exp

erim

ent


4. Check to see that current is fl owing properly - you should see bubbles forming on the two platinum electrodes.

5. After the electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads and remove the cover.

6. Remove the gel from the bed for staining.

ABOUT DNA GEL STAINING After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain® Methylene Blue. Two options are provided for using the InstaStain® Methylene Blue cards. Instructions for a third staining op-tion using liquid Methylene Blue Plus™ is also provided. Check with your instructor regarding which staining method you should use.

Method 1: One-step Staining and Destaining with InstaStain® MetBlue

Method 2: Staining with InstaStain® Methylene Blue

Method 3: Liquid Staining with Methylene Blue Plus

3. Set the power source at the required voltage and conduct electro-phoresis for the length of time determined by your instructor. General guidelines are presented in Table C.

Time and VoltageRecommendations

Minimum / Maximum

Volts

150

125

70

50

15 / 20 min

20 / 30 min

35 / 45 min

50 / 80 min

Table

CEDVOTEK Electrophoresis ModelM6+ M12 & M36

Minimum / Maximum

25 / 35 min

35 / 45 min

60 / 90 min

95 / 130 min


907EDVO-Kit #



The Exp

erim

ent

Electrophoresis - Staining and Visualization of DNA

METHOD 1: ONE-STEP STAINING AND DESTAINING WITH INSTASTAIN® METHYLENE BLUE

Agarose gels can be stained and destained in one easy step with In-staStain™ Methylene Blue cards. This one-step method can be complet-ed in approximately 3 hours, or can be left overnight.

1. Remove the 7 x 7 cm agarose gel from its bed and completely sub-merse the gel in a small, clean tray containing 75 ml of distilled or de-ionized water, or used electropho-resis buffer. The agarose gel should be completely covered with liquid.

Examples of small trays include large weigh boats, or small plastic food containers

2. Gently fl oat a 7 x 7 cm card of InstaStain® MetBlue with the stain side (blue) facing the liquid.

3. Let the gel soak undisturbed in the liquid for approximately 3 hours. The gel can be left in the liquid overnight (cover with plastic wrap to prevent evaporation).

4. After staining and destaining, the gel is ready for visualization and photography.

STORAGE AND DISPOSAL OF INSTASTAIN® METHYLENE BLUE CARDS AND GELS

• Stained gels may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid.

DO NOT FREEZE AGAROSE GELS!

• Used InstaStain® cards and destained gels can be discarded in solid waste disposal.

• Destaining solutions can be disposed down the drain.

Wear gloves and safety goggles

Do not stain gel(s) in the electrophoresis apparatus.

InstaStain™

One Step Stain and Destain

18



EDVO-Kit #


The

Exp

erim

ent

METHOD 2: STAINING WITH INSTASTAIN® METHYLENE BLUE CARDS

1. After electrophoresis, place the agarose gel on a fl at surface covered with plastic wrap.

2. Wearing gloves, place the blue dye side of the InstaStain® Methylene Blue card on the gel.

3. Firmly run your fi ngers several times over the entire surface of the InstaStain® card to establish good contact between the InstaStain® card and the gel.

4. To ensure continuous contact between the gel and the InstaStain® card, place a gel casting tray and weight, such as a small empty beaker, on top of the InstaStain® card.

5. Allow the InstaStain® Methylene Blue to sit on the gel for 5 to 10 minutes.

6. After staining, remove the InstaStain® card.

If the color of the gel appears very light, wet the gel surface with buffer or distilled water and place the InstaStain® card back on the gel for an additional 5 minutes.

Destaining and Visualization of DNA

7. Transfer the gel to a large weigh boat or small plastic container.

8. Destain with distilled water.*

• Add approximately 100 ml of distilled water to cover the gel.


InstaStain™

Patents Pending

DNA InstaStain™

Patents Pending

Patents Pending

InstaStain™

-----

1

2

3

4

5

6

Place gel on a flatsurface covered with plastic wrap.

Place the InstaStain®card on the gel.

Place a small weightfor approx. 5 minutes.

Transfer to a smalltray for destaining.

Destain with 37°Cdistilled water.

Press firmly.


InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending.


907EDVO-Kit #



The Exp

erim

ent


9. Repeat destaining by changing the distilled water as needed.

The larger DNA bands will initially be visible as dark blue bands against a lighter blue background. When the gel is completely destained, the larger DNA bands will become sharper and the smaller bands will be visible. With additional destaining, the entire background will become uniformly light blue.

10. Carefully remove the gel from the destain solution and examine the gel on a Visible Light Gel Visualization System. To optimize visibility, use the amber fi lter provided with EDVOTEK equipment.

11. If the gel is too light and bands are diffi cult to see, repeat the staining and destaining procedures.

* Destaining Notes

• Warmed distilled water at 37°C will accelerate destaining. Destaining will take longer with room temperature water.

• DO NOT EXCEED 37°C ! Warmer temperatures will soften the gel and may cause it to break.

• The volume of distilled water for destaining depends upon the size of the tray. Use the smallest tray available that will accommodate the gel. The gel should be completely submerged during destaining.

• Do not exceed 3 changes of water for destaining. Excessive destaining will cause the bands to be very light.

STORAGE AND DISPOSAL OF INSTASTAIN® METHYLENE BLUE CARDS AND GELS

• Stained gels may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid.

DO NOT FREEZE AGAROSE GELS!

• Used InstaStain® cards and destained gels can be discarded in solid waste disposal.

• Destaining solutions can be disposed down the drain.

20



EDVO-Kit #


The

Exp

erim

ent

DO NOT EXCEED 37°C !

Warmer temperatures will

soften the gel and may cause it to

break.


METHOD 3: LIQUID STAINING WITH METHYLENE BLUE PLUS™

Dilution of Methylene Blue Plus™ stain:

Dilute the 10x stain by mixing 1 part stain with 9 parts distilled or deionized water.

Staining and Destaining

1. Remove each agarose gel from its bed and totally submerse up to 6 gels in a tray containing 600 ml of diluted Methylene Blue Plus™ stain. Do not stain gel(s) in the electrophoresis apparatus.

Each group should mark their gel, such as removing a small slice, or making a small hole in a designated corner, to facilitate identifi cation after staining and destaining.

2. Stain gel(s) for a minimum of 30 minutes, with occasional agitation.

3. Destain in 600 ml of distilled water that has been warmed to 37°C.

• Completely submerse the gel(s) in 600 ml of 37°C distilled water for 15 minutes with occasional agitation. Then discard the de-staining solution

• Change the distilled water for a second destain for another 15 minutes with occasional agitation.

Bands will become visible after the second destain. You may also leave the gel(s) in destain overnight.

4. Carefully remove the gel from the destain solution and examine on a Visible Light Gel Visualization System. To optimize visibility, use the amber fi lter provided with EDVOTEK equipment.

5. If the gel is too light and bands are diffi cult to see, repeat the staining and destaining procedures.

STORAGE AND DISPOSAL OF STAIN AND GEL

• Gels stained with Methylene Blue Plus™ may be stored in the refrig-erator for several weeks. Place the gel in a sealable plastic bag with destaining liquid.

DO NOT FREEZE AGAROSE GELS.

• Stained gels which are not kept can be discarded in solid waste disposal. Methylene Blue Plus™ stain and destaining solutions can be disposed down the drain.


Do not stain gel(s) in the electrophoresis apparatus.


907EDVO-Kit #



The Exp

erim

ent

Answer the following study questions in your laboratory notebook or on a separate worksheet.

1. Why is EDTA used in DNA extraction buffer?

2. Is the DNA extracted intact? What class of enzymes degrade DNA during the extraction procedures?

3. Why do the extracted DNA samples primarily yield a single band with a small amount of migration and the appearance of a smear?

4. Can the extracted DNA be cut by restriction enzymes to give a set of specifi c bands?

Study Questions


907.060419

22

xxx907EDVO-Kit #



907.060419

23

EDVOTEK • The Biotechnology Education Company® 1.800.EDVOTEK • www.edvotek.com

FAX: 202.370.1501 • email: [email protected]

907EDVO-Kit #

Instructor’s Guide

Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fi t your specifi c set of circumstances. If you do not fi nd the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is avail-able from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).

NATIONAL CONTENT AND SKILL STANDARDS

By performing this experiment, students will learn to extract plant chromo-somal DNA, load samples and run agarose gel electrophoresis. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation. Please visit our website for specifi c content and skill standards for various experiments.

EDUCATIONAL RESOURCES

Electrophoresis Hints, Help and Frequently Asked Questions

EDVOTEK Electrophoresis Experiments are easy to perform and are de-signed for maximum success in the classroom setting. However, even the most experienced students and teachers occasionally encounter experi-mental problems or diffi culties. The EDVOTEK web site provides several suggestions and reminders for conducting electrophoresis, as well as answers to frequently asked electrophoresis questions.

Laboratory Extensions and Supplemental Activities

Laboratory extensions are easy to perform using EDVOTEK experiment kits. For laboratory extension suggestions, please check the EDVOTEK website, which is updated on a continous basis with educational activities and resources.

Safety Data Sheets can be found on our website:

www.edvotek.com/safety-data-sheets

24



EDVO-Kit #


Inst

ruc

tor’s

Gui

de

Time and VoltageRecommendations

Minimum / Maximum

Volts

150

125

70

50

15 / 20 min

20 / 30 min

35 / 45 min

50 / 80 min

Table

CEDVOTEK Electrophoresis ModelM6+ M12 & M36

Minimum / Maximum

25 / 35 min

35 / 45 min

60 / 90 min

95 / 130 min

Notes to the Instructor:

This experiment contains reagents for 10 groups.

APPROXIMATE TIME REQUIREMENTS

1. Prelab preparation and dispensing of biologicals and reagents will take approximately 1 hour.

2. Students should be able to complete the isolation of DNA and pre-pare the samples for electrophoresis in 50 minutes.

3. Gel preparation: Whether you choose to prepare the gel(s) in ad-vance or have the students prepare their own, allow approximately 30-40 minutes for this procedure. Generally, 20 minutes of this time is required for gel solidifi cation.

4. Practice Gel Loading: If your students are unfamiliar with using micro-pipets and sample loading techniques, a practice activity is sug-gested prior to conducting the experiment. EDVOTEK electrophoresis experiments contain a tube of practice gel loading solution for this purpose. Casting of a separate practice gel is highly recommended. This activity can require anywhere from 10 minutes to an entire labora-tory session, depending upon the skill level of your students.

EDVOTEK Experiment # S-44, Micropipetting Basics, focuses exclusively on the use of micropipets. Students learn and practice pipet-ing techniques by preparing and delivering various dye mixtures to a special Pipet Card™.

5. Conducting Electrophoresis: The approximate time for electrophoresis

will vary from approximately 15 minutes to 2 hours depending upon various factors. Different models of electrophoresis units will separate DNA at different rates de-pending upon its confi guration and the distance between the two electrodes. Generally, the higher the voltage applied the faster the samples migrate. However, the maximum amount of voltage signifi -cantly depends upon the design of the electrophoresis apparatus and should not exceed manufacturer's recommenda-tions. Time and Voltage recommenda-tions for EDVOTEK equipment are outlined in Table C.


907EDVO-Kit #



Instructo

r’s Guid

e

GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS This experiment features InstaStain® Methylene Blue for gel staining after electrophoresis. It is a proprietary new staining method which saves time and reduces liquid waste. EDVOTEK also offers InstaStain® Ethidium Bro-mide (InstaStain® EtBr) and Protein InstaStain® for staining Protein poly-acrylamide gels.

Two options are provided for using the InstaStain® Methylene Blue cards. Instructions for a third staining option using liquid Methylene Blue Plus™ is also provided.

Method 1: One-step Staining and Destaining with InstaStain® MetBlue

Method 2: Staining with InstaStain® Methylene Blue

Method 3: Liquid Staining with Methylene Blue Plus™

Using Method 1, agarose gels can be stained and destained in one easy step, which can be completed in approximately 3 hours, or can be left in liquid overnight. Method 2, using InstaStain® Methylene Blue cards, requires approximately 5-10 minutes for staining. DNA bands will become visible after destaining for approximately 20 minutes, and will become sharper with additional destaining. For the best photographic results, allow the gel to destain for several hours to overnight. This will allow the stained gel to "equilibrate" in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background.

Notes to the Instructor:

26



EDVO-Kit #


Inst

ruc

tor’s

Gui

de

Pre-Lab Preparations - DNA Isolation

PLANT MATERIAL, Component A Pisum sativum (pea seeds)

The DNA extraction in this experiment is optimized for using pea seedlings as starting material. From a single nursery fl at (21 x 10 x 2 inches) enough plant material can be harvested for each of the ten lab groups.

Plan to plant seeds two to three weeks before they will be used in the lab. Also allow time for the leaf tissue to dry after it is harvested. Seedlings can be grown on a windowsill, in a growth chamber, or in a greenhouse.

1. Soak the pea seeds (A) in a large beaker overnight.

2. Sow them in a layer of about 1.5 inches of wet horticulture grade vermiculite in a standard nursery fl at. The seeds can be very close together.

3. Cover the seeds with 0.5 - 1.0 inch of vermiculite and water well.

4. Cover the fl at with plastic wrap to hold in moisture while the seeds germinate.

5. Once the seedlings have begun to emerge from the vermiculite, remove the plastic wrap and keep the fl at well watered.

6. Harvest leaf tissue from shoots of seedlings and allow to air dry on ab-sorbent paper in the lab. Air drying may take several weeks, depend-ing on humidity and temperature in your lab. Consider freeze-drying the tissue is an alternative procedure.

7. Collect 1g of the air dried leaves per lab group for isolation of chro-mosomal DNA (approximately 10 g of fresh tissue should yield 1g of dried material). The tissue can be stored in a -20°C freezer until you are ready for Genomic DNA Extraction.

This experiment was tested using pea seedlings and various other specimens of plants. Generally, good results are obtained with most leaf tissue, especially peas, sunfl ower and clo-ver. Leaves from phenolic-rich plants such as rose leaves, strawberries and rhododendrons, as well as succulent plants such as aloe and cactus, are not recommended for this extraction procedure. DNA extracted from the plant tissue can be analyzed by electrophoresis on an agarose gel, but should not be expected to cut well by restriction endonucleases.


907EDVO-Kit #



Instructo

r’s Guid

e

Pre-Lab Preparations - DNA Isolation

Each student group requires:

• 0.5 g air-dried or freeze-dried pea shoots• Mortar and pestle• 10 ml Extraction Buffer (diluted A + B)• 4 ml DNA Wash Solution (diluted C)• 15 and 50 ml centrifuge tubes• Beakers• Isopropyl rubbing alcohol• 4 ml 1x TE (diluted D)• Gel loading solution• 0.8% agarose gel• 40 μl Standard Genomic DNA [50 μg/ml] (E)

PRE-LAB PREPARATION OF REAGENTS

1. Prepare Extraction buffer:

• Dilute 55 ml of Extraction Buffer (A) with 55 ml of distilled water.

• Add the entire contents of β-mercaptoethanol (B) to the diluted extraction buffer.

• Label 10 tubes or beakers and aliquot 10 ml per lab group and place on ice.

2. Prepare DNA wash solution

• Mix 7 ml of Ammonium Acetate, pH 7.5 (C) with 38 ml of 95% ETOH.

• Label tubes "DNA Wash Solution" and aliquot 4 ml per group.

3. Dilute TE Buffer

• Mix 4 ml of 10x TE (D) with 36 ml of distilled water.

• Label tubes "1x TE" and aliquot 4 ml per group.

4. Label 10 tubes "marker" and aliquot 40 μl of Standard Genomic DNA (E) for each student group.

If Extraction Buffer (A) has come out of solution, heat it to 65°C until it is completely redissolved.

28



EDVO-Kit #


Inst

ruc

tor’s

Gui

de

Pre-Lab Preparations - Electrophoresis

PREPARING AGAROSE GELS Preparing gels for electrophoresisThere are several options for preparing agarose gels for the electrophore-sis experiments:

1. Individual Gel Casting: Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment.

2. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for sharing by the class. See instructions for "Batch Gel Preparation".

3. Preparing Gels in Advance: • Gels may be prepared ahead and stored for later use. Solidi-

fi ed gels can be stored under buffer in the refrigerator for up to 2 weeks.

Do not store gels at -20°C. Freezing will destroy the gels.

• Gels that have been removed from their trays for storage, should be "anchored" back to the tray with a few drops of hot, molten agarose before placing the gels into the apparatus for electro-phoresis. This will prevent the gels from sliding around in the trays and the chambers.


907EDVO-Kit #



Instructo

r’s Guid

e

To save time, the electrophoiresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buf-fer can be used at a later time and solidifi ed agarose gel solution can be remelted.

Quantity Preparations for Agarose Gel Electrophoresis

BULK ELECTROPHORESIS BUFFER

Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D.

BATCH AGAROSE GELS (0.8%)

For quantity (batch) preparation of 0.8% aga-rose gels, see Table E.

1. Use a 500 ml fl ask to prepare the diluted gel buffer

2. Pour 3.0 grams of UltraSpec-Agarose™ into the prepared buffer. Swirl to disperse clumps.

3. With a marking pen, indicate the level of solution volume on the outside of the fl ask.

60˚C

Table

D


(ml)

DistilledWater(ml)

TotalVolume

(ml)

60 2,940 3000 (3 L)

=+

Bulk Preparation of Electrophoresis Buffer

4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution.

5. Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the fl ask in step 3.

6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed.

7. Allow the gel to completely solidify. It will become fi rm and cool to the touch after approximately 20 minutes. Then proceed with prepar-ing the gel for electrophoresis.

Note: The UltraSpec-Agarose™ kit com-ponent is often labeled with the amount it contains. In many cases, the entire contents of the bottle is 3.0 grams. Please read the label carefully. If the amount of agarose is not specifi ed or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.

Table

E

Amt ofAgarose

(g)


(ml)

DistilledWater(ml)

TotalVolume

(ml)

3.0 7.5 382.5 390

+ =+

Batch Preparation of 0.8%* UltraSpec-Agarose™


30



EDVO-Kit #


Inst

ruc

tor’s

Gui

de

Experiment Results and Analysis

GEL ELECTROPHORESIS RESULTS

( + )

( - )

1 2 3 4 5 6

The fi gure to the left is an idealized schematic showing the relative mobility of the genomic DNA samples. The idealized schematic shows the relative positions of the bands, but are not depicted to scale.

Lane 1: Intact Genomic DNA In this example, a band near the edge of the well may sometimes be observed. This extra band can be due to residual bound proteins that retard migration of the DNA into the gel.

Lane 3: Example of badly sheared Genomic DNA

Please refer to the kit insert for the Answers to

Study Questions

The Biotechnology Education Company - EDVOTEK · The Biotechnology Education Company ... • Mortar...

Documents

Transcript of The Biotechnology Education Company - EDVOTEK · The Biotechnology Education Company ... • Mortar...