Bio-Rad 12016408EDU User guide

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BIO-RAD

DNA Extraction Module

Catalog #12016408EDU

Instruction Manual

Note: Duplication of any part of this document is permitted for noncommercial, educational use only. Commercial use of copyrighted content, including use in instructional materials for which payment is received, requires express permission from Bio-Rad Laboratories, Inc. Contact us at [email protected] for more information.

For technical support, call your local Bio-Rad ofﬁce, or in the U.S. call 1-800-4BIORAD (1-800-424-6723) option 2.

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Table of Contents

Before You Start

Storage ..................................................................................................................................................... 1

Safety Guidelines ....................................................................................................................................... 1

Components ............................................................................................................................................. 2

Ordering Information .................................................................................................................................. 3

Instructor Guide

Preparation Instructions ............................................................................................................................. 4

Tips and Notes ......................................................................................................................................... 6

Student Guide

Materials Required ..................................................................................................................................... 7

Protocol ..................................................................................................................................................... 7

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Before You Start

–20°C

500 ml

Agar

00:00

Storage

When you receive the DNA Extraction Module:

Note storage location and record the batch

numbers from the product labels.

Store the DNA Extraction Module at room

temperature.

Visit bio-rad.com/DNAExtractionMod to

download the most up-to-date instruction manual.

Technical Support is available at [email protected] or

1-800-4BIORAD, option 2.

Room Temp

~20°C

Safety Guidelines

Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Wearing protective eyewear and gloves is strongly recommended. Students should wash their hands with soap before and after this exercise. If any solution gets into a student’s eyes, ﬂush with water for 15 min. Lab coats or other protective clothing should be worn to avoid staining clothes.

Always use proper safety precautions when collecting and processing samples. Some organisms are harmful and/or poisonous if handled improperly. Improper handling may result in serious injury or death. It is recommended that students work only with samples that have been positively identiﬁed as nontoxic, or otherwise use samples from a reliable retailer such as a grocery store, pet store, or science supply company. Use appropriate personal protective equipment minimally including gloves and safety goggles. Use extreme caution when using cutting utensils to prevent injury. Use standard microbiology safety practices when working with samples that may contain pathogenic bacteria. Place any materials that contacted bacteria in a 10% bleach solution for at least 20 min to decontaminate. Follow local regulations for further disposal recommendations.

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Visit explorer.bio-rad.com to access Safety Data Sheets for individual products.

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Before You Start

Components

The DNA Extraction Module includes materials for 16 samples, or

32 students working in pairs (one sample each) or groups of four

(two samples each).

Materials included in this module

Item Quantity

DNA Extraction Module

Resuspension solution 5 ml Lysis solution 5 ml Neutralization solution 5 ml Matrix 5 ml Wash buffer 10 ml Spin columns 20 2 ml capped microcentrifuge tubes 250

Required materials not included in this module

Item Quantity

Equipment

100–1,000 μl adjustable-volume micropipet and tips 8–16 Dry bath or water bath 1 Microcentrifuge, ≥14,000 x g 1 Inoculation loop, sterile, if using bacterial samples 16 Razor blade or plastic knife, new and unused, if using 16 tissue samples Weigh boat 16 Microcentrifuge tube rack 8–16 Permanent marking pen 8–16

Reagents and Consumables

Bulk tissue samples or bacteria culture 1–16 Distilled water 3 ml Ethanol, 95% 10 ml Bleach solution, 10%

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Ordering Information

Catalog # Description

Kits and Reﬁll Packs

12016408EDU DNA Extraction Module 1665105EDU DNA Extraction Reagent Pack

Reagents and Consumables

2239430EDU EZ Micro Test Tubes, 500 1660471EDU Inoculation Loops, Sterile, 100

Equipment and Laboratory Supplies

1660506EDU Professional Adjustable-Volume Micropipet, 2–20 μl 1660507EDU Professional Adjustable-Volume Micropipet, 20–200 μl 1660508EDU Professional Adjustable-Volume Micropipet, 100–1,000 μl 1660562EDU Digital Dry Bath, 120 V 1660504EDU Temperature-Controlled Water Bath, 120 V 12011919EDU Mini Centrifuge, 100–240 V 1660602EDU Model 16K Microcentrifuge, 120 V 1660610EDU BR-2000 Vortexer, 120 V 1660481EDU Green Racks, set of 5

Before You Start

Visit explorer.bio-rad.com for a full list of 220–240 V equipment.

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Instructor Guide

Preparation Instructions

1. Use Table 1 to prepare and dispense solutions up to four weeks before the activity.

To help prevent cross-contamination, label tubes and dispense one solution at a time. Be sure to use a new pipet tip for each solution.

Table 1. Reagent preparation and dispensing instructions.

Tube Type and Quantity

16 microcentrifuge tubes with caps, 2.0 ml

2. Acquire tissue samples (if using) any time before the lessons.

Source tissue samples or have your student source tissue samples any time before the activity. To avoid spoilage and

contamination, store tissue samples frozen and then thaw the day of the activity. Plant tissue samples are not recommended for

this DNA extraction protocol.

Tube Label

R 250 μl resuspension solution Up to 4 weeks

Lys 300 μl lysis solution Up to 4 weeks

N 300 μl neutralization solution Up to 4 weeks

Wash

dH2O

Tube Contents and Preparation Instructions Storage

at room temp.

250 μl resuspended matrix

Preparation: Before dispensing, resuspend the resin matrix by shaking, stirring manually, or vortexing

1 ml wash buffer plus ethanol solution

Preparation: Before dispensing, add 10 ml of 95% ethanol to the bottle of wash buffer, replace the cap, and shake to mix.

150 µl distilled water

Up to 4 weeks at room temp.

1–2 days at room temp.

Indeﬁnitely

Table 2. Samples, by species, that reliably produce successful results.

Robust

Insects

Beetles Moths

Mammals and Birds

Cow Pig

Fungi

Fresh mushroom caps Dried mushrooms are less robust

Fish

Dried ﬁsh Catﬁsh Shark Sturgeon Trout Rock cod

Butterﬂies Bees

Sheep Deer

True cod Tilapia Mackerel Yellowﬁn tuna Sea bass Opah

Wasps Flies

Pheasant Duck

Imitation crab Flying ﬁsh roe Bonito ﬂakes Shrimp

Less Robust

Spiders Molted exoskeletons

Elk Turkey

than fresh mushrooms, but can still produce good results

Salmon Salmon roe Anchovy Arctic char Sardine

Difﬁcult/Not Robust

Cocoons

Ground meat Fur

Older mushrooms, due to the risk of contamination

Fried ﬁsh Canned ﬁsh Sea urchin Red tuna Mussel Clam

Gastropods

Feathers Bones

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Instructor Guide

3. Prepare bacteria cultures (if using) 1–5 days before the DNA extraction activity.

For each student sample, prepare a bacterial culture plate or 3 ml of liquid bacterial culture from a single colony. Chromosomal

DNA can be extracted from either bacterial colonies or liquid culture.

4. Prepare student workstations.

Use Tables 3–4 to set up workstations just before the activity. Each workstation has enough materials to extract DNA from one

sample. Micropipets can be shared if necessary between groups. Other materials should not be shared,to avoid contamination.

Table 3. Student workstation.

Materials Quantity

Resuspension buffer (R)

Lysis buffer (Lys)

Neutralization buffer (N)

Matrix (X)

Wash buffer (Wash)

Distilled water (dH2O)

Empty 2 ml microcentrifuge tubes with caps

Empty 2 ml microcentrifuge tubes with caps removed

Spin column

Bulk tissue sample or bacterial culture

Empty, clean weigh boat

Inoculation loop, sterile, if using bacterial samples

(Note: It is critical to use one loop per sample)

Razor blade, plastic knife, or other clean cutting utensil, if using tissue samples

(Note: it is critical to use one utensil per sample)

100–1,000 µl adjustable-volume micropipet and tips

Marking pen

Bleach solution, 10%

Table 4. Common workstation.

250 µl

300 µl

250 µl

1 ml

250 µl

Materials Quantity

Water bath or dry bath set to 55°C

Microcentrifuge

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1–2

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Instructor Guide

of contamination.

Tips and Notes

The success of many downstream analyses, including polymerase chain reaction (PCR), is highly dependent on the quality and

quantity of DNA extracted from your samples. Use the following tips to ensure successul DNA extraction.

Preventing contamination is of utmost importance. Use the following precautions to minimize the chance

of contamination.

• New, clean, and DNA-free cutting utensils are required for each sample. Do not cross-contaminate samples. The DNA

from a single contaminating cell can ruin results. Use gloves and change them between specimens

• Wiping a utensil and rinsing in ethanol between samples will not sufﬁ ciently remove DNA present on a cutting utensil

• When working with non-bacterial samples, it is possible for DNA from contaminating bacteria to coamplify with sample DNA.

Taking samples from the interior of bulk animal tissue can reduce the chances of contamination with bacteria

• When selecting samples, avoid fur, feathers, exoskeleton, bones, or other keratinous or chitinous samples. Also avoid

canned or processed samples, as acidic conditions can damage DNA. High fat–content samples (for example, fried foods)

may also inhibit DNA extraction and subsequent PCR. Fresh, frozen, or dried samples can produce excellent results

• Once lysis buffer is added during DNA extraction, there is no convenient stopping point until the DNA is eluted from the spin

puriﬁ cation column. If you choose to stop before adding the lysis buffer, be sure student samples are labeled with their initials,

and store them refrigerated at 4°C for up to 1 week

• Wipe down surfaces and rinse plastic pipet barrels, mortars, and pestles with 10% bleach to remove any surface DNA

contamination.

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Materials Required

Student Workstation

Materials Quantity

Student Guide

Resuspension buffer (R)

Lysis buffer (Lys)

Neutralization buffer (N)

Matrix (X)

Wash buffer (Wash)

Distilled water (dH2O)

Empty 2 ml microcentrifuge tubes with caps

Empty 2 ml microcentrifuge tubes with caps removed

Spin column

Bulk tissue sample or bacterial culture

Empty, clean weigh boat

Inoculation loop, sterile, if using bacterial samples

(Note: It is critical to use one loop per sample)

Razor blade, plastic knife, or other clean cutting utensil

(Note: It is critical to use one utensil per sample)

100–1,000 µl adjustable-volume micropipet and tips

Marking pen

Bleach solution, 10%

Common Workstation

Materials Quantity

250 µl

300 µl

250 µl

1 ml

150 µl

Water bath or dry bath set to 55°C

Microcentrifuge

1–2

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Student Guide

Protocol

1. Label one capped 2 ml microcentrifuge tube with your initials.

If provided or known, record the ID or a description

of the tissue sample you will be using.

Sample __________________

2. Prepare your sample by following the steps below that correspond to your sample type.

For bacteria samples on solid media

a. Add 200 µl resuspension buffer (R) to the

labeled 2 ml microcentrifuge tube.

b. Use a new sterile pipet tip or inoculating loop to

pick one bacterial colony from a culture plate and transfer to the tube.

➜

c. Completely resuspend the colony in the

resuspension buffer by pipetting up and down or vortexing for 10–20 sec.

For bacteria samples in liquid culture

a. Transfer 1–2 ml fresh overnight bacteria liquid

culture to the labeled 2 ml microcentrifuge tube.

b. Centrifuge the sample at 14,000 x g for 30 sec

to form a cell pellet. Be sure to use a counterbalance.

➜

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Student Guide

c. Use a micropipet to carefully remove the

supernatant without disturbing the pellet. Discard the supernatant into a 10% bleach solution.

d. Add 200 µl of resuspension buffer (R) and pipet

up and down or vortex to completely resuspend the bacteria.

For ﬁ sh, mammalian, bird, or fungal samples

a. Cut a piece of tissue sample up to 100 mg,

approximately the size of an eraser-head, from the bulk tissue sample and place it directly into the appropriately labeled 2 ml microcentrifuge tube.

➜

Note: Cut a sample from the interior of the

bulk tissue to avoid bacterial contamination that may be present on the surface. Avoid fur, scales, or feathers.

b. Add 200 µl resuspension buffer (R).

c. Take a micropipet tip in hand and ﬁ rmly press

the narrow end at a slight angle into the sample weigh boat or a new weigh boat to bend and crush a small portion of the tip.

d. Use the crushed micropipet tip as a pestle to

grind the sample in the microcentrifuge tube for at least 30 sec until the sample becomes cloudy. Then discard the tip.

➜

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Student Guide

For insect samples

a. Cut a small piece of tissue approximately half

the size of an eraser-head from the abdomen of your insect. The total mass will vary. Place the piece directly into the appropriately labeled microcentrifuge tube.

Note: Avoid wings, legs, and other chitinous parts of the insect body, as they are much less effective for DNA extraction.

b. Add 200 µl resuspension buffer (R).

c. Take a micropipet tip in hand and ﬁ rmly press

the narrow end at a slight angle into the sample weigh boat or a new weigh boat to bend and crush a small portion of the tip.

➜

d. Use the crushed micropipet tip as a pestle to

grind the sample in the microcentrifuge tube for at least 30 sec until the sample becomes cloudy. Then discard the tip.

Stop. Ask your instructor whether to proceed now or later.

3. Add 250 µl lysis buffer (Lys) and mix gently by inverting each tube 10 times. DO NOT VORTEX. Vortexing may shear genomic DNA, which can inhibit PCR ampliﬁ cation.

4. Incubate sample at 55°C for 10 min in a water bath or dry bath.

➜

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Student Guide

5. Add 250 µl neutralization buffer (N) and mix gently by inverting 10 times. DO NOT VORTEX. A cloudy precipitate may form.

6. Centrifuge for 5 min at 12,000–14,000 x g. Be sure to use a counter balance. A compact pellet will form along the side of the tube. The supernatant contains the DNA.

If there are many particulates in the supernatant

after centrifugation, centrifuge the tubes for an additional 5 min.

7. Snap off (do not twist!) the bottom of the spin column. Insert the column into a capless 2 ml microcentrifuge tube.

➜

8. Label the spin column with your initials.

9. Using a new pipet tip for each sample, transfer ~500 µl supernatant from each sample to the appropriately labeled spin column.

Do not disturb the tissue pellet.

10. Vortex or repeatedly shake the matrix (X) to make sure the resin is completely resuspended.

11. Quickly add 200 µl thoroughly resuspended matrix (X) to the spin column. Pipet up and down to mix.

12. Centrifuge the spin column with the capless microcentrifuge tube at full speed (14,000 x g)

for 30 sec. Be sure to use a counter balance.

➜

Centrifuge for only 30 sec! Completely drying the matrix at this point will result in DNA loss.

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13. Remove the spin column from the capless 2 ml microcentrifuge tube, discard the ﬂ owthrough, and replace the spin column into the same tube.

14. Add 500 µl wash buffer (Wash) and wash the matrix by centrifuging for 30 sec. Be sure to use a counterbalance.

Centrifuge for only 30 sec! Completely drying the matrix at this point will result in DNA loss.

15. Remove the spin column from the capless 2 ml microcentrifuge tube, discard the ﬂ owthrough,

and replace the spin column into the same tube.

16. Finally, centrifuge the spin column with the

capless microcentrifuge tube at full speed for a full 2 min to remove residual traces of ethanol and dry out the matrix. Be sure to use a

counterbalance.

17. Label a clean 2 ml capless microcentrifuge tube with your initials.

➜

18. Transfer the spin column into the appropriately labeled new, clean capless microcentrifuge tube. Discard the used capless microcentrifuge tube along with any liquid in the tube.

19. Using a new pipet tip, add 100 µl distilled water to the spin column, being careful not to touch the resin.

20. Centrifuge the spin column with the capless microcentrifuge tube at full speed (14,000 x g)

for 1 min. Be sure to use a counterbalance.

➜

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21. Label a new 2 ml capped microcentrifuge tube with your initials.

➜

22. Remove the spin column from the capless microcentrifuge tube and transfer the ﬂ owthrough which contains eluted DNA, into the appropriately labeled 2 ml capped microcentrifuge tube.

➜

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