Bio-Rad 732-6212 User guide

Bio-Rad
Laboratories

®

mD

Product Line

x

InstaGene™ Dry Blood Kit

Instruction Manual

Catalog Number 732-6212

125 DNA preparations from 1/8”-diameter dry blood spots

Please familiarize yourself with the contents of this insert before using the product for the first time.

1. INTRODUCTION

The InstaGene Dry Blood Kit is used to purify genomic DNA from blood that has been spotted onto filter paper (e.g.,
Schleicher and Schuell #903™ filter paper). The 45 minute procedure results in the selective extraction of amplification
reaction inhibitors present in blood without removing any of the filter-bound DNA. The extracted dry blood spot (DBS) is
added directly to the amplification reaction mixture prior to thermocycling.

2. KIT COMPONENTS AND STORAGE

Component Description Amount

Extraction Reagent

Wash Reagent

3. ITEMS REQUIRED BUT NOT PROVIDED

• 1.5 ml microcentrifuge tubes (Cat. No. 223-9501).

• Disposable fine-tip transfer pipettes (Cat. No. 223-9528).

• Micropipette tips, 20-200 µl and 100-1000 µl (Cat. No. 211-2016 and 211-2021, respectively).

• Paper punch, 1/8” or 1/4” punch diameter

• Adjustable micropipettes, 20-200 µl and 100-1000 µl

• Sterile distilled or deionized water

• 95% or absolute ethanol

• 65oC heat block (or 65oC water bath)

• Vortex mixer

• Disposable gloves

• SpeedVac™ concentrator (recommended)

Solution of ethanol and
chaotrope. Flammable.

Solution of ethanol and
salts. Flammable.

Available from Bio-Rad

Not available from Bio-Rad

25 ml 15-30 oC

50 ml 15-30 oC

Storage

Temperature

page 1

Bio-Rad
Laboratories

4. SAFETY

4.1 The Centers for Disease Control recommend the use of “universal precautions” when handling blood and certain

body fluids. Under universal precautions, blood and certain body fluids of all patients are considered potentially
infectious for Hepatitis B Surface Antigen (HbsbAg), HIV-1/2, HTLV-1/2, HCV, HBV and other bloodborne
pathogens. It is recommended that workers wear protective barriers such as gloves, gowns, aprons, masks or
protective eyewear to reduce the risk of exposure of the worker’s skin or mucous membranes to potentially infective
materials.

4.2 Kit reagents contain potentially harmful substances, including ethanol and guanidinium thiocyanate (See Product
Safety Information, page 4). Please refer to the Material Safety Data Sheets for instructions on proper handling and
disposal of these reagents.

5. SUMMARY OF THE PROCEDURE

Place DBS in a 1.5 ml tube.
Add 1 ml water.

Wash 1

Incubate 10 minutes at 15-30oC. Shake
occasionally.
Remove and discard supernatant.
Repeat for a total of 2 times.
Add 100 µl Extraction Reagent.

Extraction

Incubate 10 minutes at 65oC. Mix occasionally.
Remove and discard supernatant.
Repeat for a total of 2 times..
Add 100 µl Wash Reagent.

Wash 2

Vortex briefly.
Remove and discard supernatant.
Repeat for a total of 3 times.
Add 100 µl ethanol.
Vortex briefly.Wash 3
Remove and discard the supernatant.

Drying

Evaporate off ethanol (SpeedVac concentrator,
10 minutes; or air dry, at least 30 minutes).

page 2

Bio-Rad
Laboratories

6. EXTRACTION PROCEDURE

6.1 Place the DBS sample (one 1/8”-punch) in a 1.5 ml microcentrifuge tube. Add 1 ml of water. Incubate at 15-30oC for
10 minutes with occasional inversion of the tube. Pipet off the supernatant and discard.

6.2 Repeat Step 6.1.

6.3 Add 100 µl Extraction Reagent. Incubate at 65oC for 10 minutes with occasional mixing. Pipet off the supernatant,

including any liquid that might have condensed inside the tube cap, and discard.

6.4 Repeat Step 6.3. The filter should appear slightly brown or white after the second extraction step.

6.5 Add 100 µl Wash Reagent. To ensure that all the inside surfaces of the tube are washed, vortex for 1 second while

the tube is in the upright position, then vortex again for 1 second while the tube is in the inverted position (make
sure the tube is tightly capped!). Pipet off the supernatant, including any liquid inside the cap, and discard.

6.6 Perform Step 6.5 two more times.

6.7 Add 100 µl ethanol. Vortex as in Step 6.5. Pipet off the supernatant, including any liquid inside the cap, and discard.

6.8 Place the tube containing the filter in a SpeedVac concentrator and dry the filter under vacuum for 10 minutes.
Alternatively, air dry the filter for 30 minutes or longer.

7. AMPLIFICATION REACTION PROCEDURE

7.1 A typical amplification reaction will consist of one-half of an extracted 1/8” punch in a 25 µl reaction volume, or the
whole 1/8” punch in a 50 µl reaction volume.

7.2 Amplification reaction conditions have to be optimized for specific applications. Conditions found suitable for DNA
templates in solution usually apply without further modification when using filter-bound DNA template prepared with
this kit.

7.3 Make sure that the filter paper is completely immersed in the reaction mixture.

8. TROUBLESHOOTING GUIDE

DNA prepared using the InstaGene Dry Blood Kit is PCR-ready. However, there are two common causes for low
amplification yield.

8.1 Incomplete removal of the Extraction Reagent. The Extraction Reagent contains a chaotrope that if carried over to the
amplification reaction can result in a low yield. Perform the post-extraction step washes as described in Step 6.5.

8.2 Incomplete removal of ethanol after the last wash step. To obtain optimal yield, it is necessary that all traces of
ethanol be removed from the filter. Dry the filter completely as described in Step 6.8.

page 3

Bio-Rad
Laboratories

9. PRODUCT SAFETY INFORMATION

Extraction Reagent

R11, 32
S7/9, 16

Risk and Safety Phrases:

R11 Highly flammable.
R32 Contact with acid liberates very toxic gas.
S7/9 Keep container tightly closed and in a well-ventilated place.
S16 Keep away from source of ignition - No smoking.

Wash Reagent

R11
S7/9, 16

Bio-Rad Phone Numbers: United States 1-800-224-6723 • Australia 61-2-9914-2800 • Austria 43-1-877-8901 • Belgium 32-9-

385-5511 • Canada 905-712-2771 • China 86-10-6205-1850 • Denmark 45-39-17-99-47 • Finland 358-9-804-22-00 • France 33-1­4390-4690 • Germany 49-89-31884-0 • Hong Kong 852-2789-3300 • India 91-11-460-3676 • Israel 972-3-9514127 • Italy 39-02-
216091 • Japan 81-3-5811-6290 • Korea 82-2-3473-4460 • Latin America 305-894-5950 • Mexico 52-5-514-0497 • The
Netherlands 31-318-540666 • New Zealand 64-9-443-3099 • Russia 7-095-4585822 • Singapore 65-272-9877 • Spain 34-1-661­7085 • Sweden 46-8-627-50-00 • Switzerland 41-1-809-55-55 • United Kingdom 44-181-328-2000

© Copyrighted July, 1999

page 4

Bio-Rad
Laboratories

Bio-Rad Laboratories Diagnostics Group

Variant Gene 1 Instruction Manual page 5 061:001