Instruction Manual: Quick-DNA

INSTRUCTION MANUAL
Quick-DNA™ Miniprep Plus Kit

Catalog Nos. D4068 & D4069
Highlights
• Extract high-quality DNA easily and reliably from any biological fluids, cultured/monolayer cells,
or solid tissues.
• Zymo-Spin™ Technology ensures DNA is ready for all sensitive downstream applications such
as qPCR, DNA-sequencing, arrays, and methylation analysis.
Contents
Product Contents & Specifications ……………………………………………1
Sample Sources ……………………………………………………………….. 2 – 3
Product Description ……………………………………………………………. 4
Purification Guide ……………………………………………………………… 5
Protocol & Reagent Preparation ……………………………………………... 6
Appendices
A. Cell Monolayer/Buccal Cell Collection and Preparation …….. 7
B. Samples in DNA/RNA Shield™ …………………….……………8
C. Nucleated Blood Samples …………………………………..…. 9
D. Hair and Feathers …………………………………………….…. 10
E. FFPE Tissue ……………………………………………………... 11
F. Samples Collected onto Storage Papers/Cards ………………12
G. Quick Protocol ………………………………………………….... 13 – 14
Troubleshooting ………………………………………………………………... 15 – 17
Ordering Information …………………………………………………………... 18
For Research Use Only Ver. 1.3.1
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Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ info@zymoresearch.com ▪ www.zymoresearch.com
Page 1
Satisfaction of all Zymo

Research products is
Product Contents
guaranteed. If you are not
satisfied with this product Quick-DNA™ Miniprep Plus Kit D4068 D4069 Storage
please call 1-888-882-9682. (Kit Size) (50 Preps.) (200 Preps.) Temperature
Proteinase K & Storage Buffer 20 mg 4 x 20 mg −20°C (after mixing)
BioFluid & Cell Buffer (Red) 12 ml 45 ml Room Temp.

Solid Tissue Buffer (Blue)* 6 ml 22 ml Room Temp.
Genomic Binding Buffer 25 ml 85 ml Room Temp.
DNA Pre-Wash Buffer * 30 ml 2 x 50 ml Room Temp.
g-DNA Wash Buffer 50 ml 200 ml Room Temp.
DNA Elution Buffer 10 ml 50 ml Room Temp.
Zymo-Spin™ IIC-XLR Columns 50 columns 200 columns Room Temp.

Collection Tubes 100 tubes 400 tubes Room Temp.
Instruction Manual 1 1 -
Note - Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely
tested on a lot-to-lot basis to ensure they provide maximal performance and reliability.
* The Solid Tissue Buffer (Blue) and DNA Pre-Wash Buffer may have formed a precipitate. If this is the case,
incubate at 37oC to solubilize. DO NOT MICROWAVE.
Specifications
• Sample Sources – See pages 2 and 3.
• Workflow Overview – Utilizes a Proteinase K Digestion and Zymo-Spin™ Technology for
effective recovery of DNA. See page 5 for more information.
• DNA Types – The Quick-DNA™ Miniprep Plus Kit will isolate total DNA including genomic,
mitochondrial, plasmid, viral, parasitic, etc. from biological fluids, cultured/monolayer cells, or
solid tissues. Not recommended for small, cell-free DNA isolation from urine and serum/plasma
(see specialized kits D3061 & D4076 respectively).
• DNA Purity - High quality DNA is ready for all sensitive downstream applications such as PCR,
endonuclease digestion, Southern blotting, genotyping, Next-Generation Sequencing, bisulfite
conversion, etc. (A260/A230 ≥ 2.0).
• DNA Size - Capable of recovering genomic and mitochondrial DNA sized fragments ˃ 50 kb. If
present, parasitic, microbial, and viral DNA will also be recovered.
• DNA Yield - The DNA binding capacity of the column is 25 µg. Typically, mammalian tissues
yield: 1-3 µg DNA per mg skeletal, heart, lung, and brain tissues and 3-5 µg DNA per mg liver
and kidney. Human whole blood will yield 3-7 µg DNA per 100 µl blood sampled.
• Elution Volume - DNA can be eluted into as little as 35 µl DNA Elution Buffer or water.
Note - ™ Trademarks of Zymo
Research Corporation. This product • Equipment - Water bath or heat block (55˚C), microcentrifuge, and vortex.
is for research use only and should
only be used by trained • DNA Applications – DNA isolated using the Quick-DNA™ Miniprep Plus Kit can be used for
professionals. It is not intended for
use in diagnostic procedures. Some life-science research, genotyping, livestock breeding, veterinary research, and routine applied
reagents included with this kit are testing among a variety of other applications.
irritants. Wear protective gloves and
eye protection. Follow the safety
guidelines and rules enacted by
your research institution or facility.
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Sample Sources
Biological Fluids: For total DNA isolation from ≤ 200 µl of whole blood, nucleated blood,
buffy coat, saliva, sputum, semen, milk, etc.
Special Considerations
• For biological fluids samples stored in DNA/RNA Shield ™, see Appendix B (pg. 8).
• For nucleated blood samples, such as avian blood, see Appendix C (pg. 9).
• For blood, saliva, and cells collected onto Guthrie, FTA ®, and other storage
papers (cards), see Appendix F (pg. 12)
• For viral DNA isolation from serum/plasma samples, follow the Biological Fluids
& Cells workflow. For small, cell-free DNA isolation from serum/plasma, use the
Quick-cfDNA™ Serum & Plasma Kit (D4076).
• To isolate cellular and/or cell-free DNA from up to 40 ml of urine samples, see

the Quick-DNA™ Urine Kit (D3061). For cellular DNA from urine, pellet at
3,000 x g for 15 minutes and remove supernatant before processing using the
Biological Fluids & Cells workflow.
Mammalian/Insect Cell Cultures: For total DNA isolation from ≤ 5 x 106 cells such as
HeLa cells, HEK-293 cells, Drosophila cell lines, etc.
• Media should be removed before processing by pelleting cells (at approximately

500 x g for 2 minutes depending on volume and cell type) and removing the
supernatant.
• For mammalian cell samples, it is possible to reduce Proteinase K digestion time

to 5 minutes at 55˚C (Step 2 on pg. 6).
• For cell monolayer and buccal cell preparation and collection, see Appendix A.
(pg. 7).
• For samples stored in DNA/RNA Shield™, see Appendix B (pg. 8).
Bacterial Cell Cultures: For total DNA isolation (e.g. genomic, plasmid, etc.) from
≤ 5 x 106 E. coli cells.
• Media should be removed before processing by pelleting cells (at approximately

500 x g for 2 minutes depending on volume and cell type) and removing the
supernatant.
• For E. coli samples, follow the Biological Fluids & Cells workflow. All other
bacterial samples may be resistant to chemical lysis and Proteinase K digestion
and should be processed using the ZymoBIOMICS™ DNA Miniprep Kit (D4300).
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Solid Tissues: For total DNA isolation from ≤ 25 mg tail snips, ear punches, organ biopsies
(brain, liver, heart, kidney, muscle, stomach, bladder, intestine, etc.).
• Overnight Proteinase K digestion at 55˚C is possible (Step 2, pg. 6).
• For solid tissue samples stored in DNA/RNA Shield™, see Appendix B (pg. 8).
• For hair and feather samples, see Appendix D (pg. 10).
• For FFPE samples, see the Quick-DNA™ FFPE Kit (D3067) for specialized FFPE
DNA purification. See Appendix E (pg. 11) for an adapted protocol using the
Quick-DNA™ Miniprep Plus Kit.
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Product Description
The Quick-DNA™ 96 Plus Kit
The Quick-DNA™ Miniprep Plus Kit is the easiest method for high yield total (D4070, D4071) provides
DNA extraction (e.g., genomic, plasmid, mitochondrial, viral) from any biological fluid, high-throughput (i.e., 96-well
plate) processing of biological
cell culture, or solid tissue sample. Innovative reagents and Zymo-Spin™ Technology fluid, cell culture, and solid
allow for ultra-pure and concentrated genomic DNA ˃ 50 kb to be eluted in as little as tissue samples.
35 µl. Zymo-Spin™ Columns ensure no buffer retention. Purified DNA is RNA-free,
bypassing the need for RNase A treatment and ensuring accurate quantification for
applications like library preparations. Isolated DNA is suitable for immediate use in
sensitive downstream applications including qPCR, DNA-seq, arrays, and For routine plasmid DNA
methylation analysis. purification from E. coli, Zymo
Research offers the Zyppy™
Plasmid Miniprep Kit
(D4036) and the
ZymoPURE™ Midi, Maxi, and
Gigaprep Kits (D4200,
M Human Porcine HeLa Buccal Human Mouse Mouse Mouse Bovine Bovine M D4202, and D4204).
Blood Blood Cells Swab Saliva Tail Kidney Brain Muscle Milk
Zymo Research offers the EZ

DNA Methylation-Lightning™
Kit (D5030, D5031) for rapid,
precise DNA methylation
detection and a
comprehensive selection of
other epigenetic tools.
High Quality DNA Obtained from a Wide Range of Biological Samples Using the Quick-DNA™ Miniprep
Plus Kit. DNA purified using the Quick-DNA™ Miniprep Plus Kit is ultrapure, highly concentrated, and ready for all
downstream applications. Input DNA was standardized to 300 ng and analyzed in a 1% (w/v) TAE/agarose/EtBr
gel (shown above). The size marker “M” is a 1 kb ladder (Zymo Research).
Looking to isolate RNA? For
RNA isolation from TRIzol®,
the Direct-zol™ RNA
Miniprep Plus Kits (R2070,
R2072, R2071, R2073) offer
total RNA purification
without phase separation in
only 7 minutes!
DNA Yields Increase Linearly with Increasing

HSV-1 Viral DNA is Effectively Isolated from
Volumes of Human Whole Blood Using the
Plasma Using the Quick-DNA™ Miniprep Plus Kit.
Quick-DNA™ Miniprep Plus Kit. Six replicates of
A dilution series of HSV-1 spiked into porcine
25, 50, 100, and 200 µl of human whole blood were
plasma and extracted using the Quick-DNA™
processed.
Miniprep Plus Kit shows effective purification and
subsequent qPCR amplification, even at a 20,000:1
dilution. The no template controls did not amplify
even after 50 cycles.
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Purification Guide
The Quick-DNA™ Miniprep Plus Kit facilitates rapid and efficient purification of DNA from
any biological fluids, cultured/monolayer cells, or solid tissues by combining enzymatic and
chemical extraction regimens.

Workflow
Biological Fluids & Cells Solid Tissues

Biological Fluids: ≤ 200 µl
* Viral DNA from serum or
Whole blood, nucleated blood, semen, Solid Tissues: ≤ 25 mg
plasma samples can also be buffy coat, saliva, body fluids, milk, etc.* Tail snips, ear punches, organ biopsies
processed using this (Brain, liver, heart, kidney, muscle,
workflow. Not recommended Blood, saliva, and cells collected on stomach, bladder, intestine, etc.).
for cell-free DNA isolation
from urine, serum, or plasma
storage paper/cards (Appendix F).
samples. FFPE samples (Appendix E).
For cell-free DNA isolation
Cultured Cells: ≤ 5 x 106
E. coli, insect, or mammalian cells (e.g. Hair and feather samples (Appendix D).
from up to 40 ml urine, see
the Quick-DNA™ Urine Kit HeLa cells, buccal cells, HEK-293 cells,
(D3061). For cell-free DNA Drosophila cells, etc.).
isolation from up to 10 ml
serum or plasma samples,
see the Quick-cfDNA™
Serum & Plasma Kit
(D4076).
BioFluid & Cell Buffer Solid Tissue Buffer

(Red) (Blue)
Proteinase K Digestion
at 55ºC
Genomic Binding
Buffer
Spin
™
Wash Zymo-Spin
Elute Technology
Ultra-Pure DNA
ZYMO RESEARCH CORP.
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Notes:
Reagent Preparation 1
If using ˂ 50 µl sample,
✓ Add 1,040 µl Proteinase K Storage Buffer to each Proteinase K (20 mg) tube prior to increase the volume to 50 µl
using DNA Elution Buffer or
use. The final concentration of Proteinase K is ~20 mg/ml. Store at -20ºC after mixing. an isotonic buffer (PBS)
before continuing.
Protocol
Resuspend cultured cell or E. coli pellets using DNA Elution Buffer or an isotonic buffer (e.g. PBS):
˂ 1 x 106 cells in 100 µl
1-5 x 106 cells in 200 µl
Overnight Proteinase K digestions at 55ºC are possible without affecting the integrity of the DNA.
2
Avoid transferring lipid layer
and pelleted cellular debris.
Biological Fluids & Cells Solid Tissues
1. Add up to 200 µl sample to a 1. To a tissue sample (≤ 25 mg) in a
microcentrifuge tube and add: microcentrifuge tube, add a
solution of:
200 µl BioFluid & Cell Buffer (Red)
20 µl Proteinase K 95 µl Water
3
95 µl Solid Tissue Buffer (Blue) If the lysate is still visible on
Note: For inputs ˂ 200 µl biological fluid,
1
10 µl Proteinase K top of the matrix, centrifuge
proportionally decrease BioFluid & Cell Buffer for another minute or until
(Red), Proteinase K, and Genomic Binding completely cleared.
Buffer. 2. Mix thoroughly or vortex 10-15
seconds and then incubate the
tube at 55ºC for 1-3 hours or until
2. Mix thoroughly or vortex 10-15 tissue solubilizes. Mix thoroughly
seconds and then incubate the tube before proceeding.
at 55ºC for 10 minutes. Note: To remove insoluble debris, centrifuge at
≥ 12,000 x g for 1 minute. Transfer aqueous 4
You can elute in as little as
supernatant2 to a clean microcentrifuge tube. 35 µl for highly concentrated
DNA. See Figure on pg. 16
for more information.
3. Add 1 volume Genomic Binding 3. Add 2 volumes Genomic Binding
Buffer to the digested sample. Mix Buffer to the supernatant. Mix
thoroughly or vortex 10-15 thoroughly or vortex 10-15
seconds. seconds.
Example: Add 420 µl Genomic Binding Example: Add 400 µl Genomic Binding
Buffer to the 420 µl digested sample. Buffer to the 200 µl supernatant.
5
DNA Elution Buffer:
10 mM Tris-HCl, pH 8.5, 0.1
4. Transfer the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube. mM EDTA. If water is used,
ensure the pH is > 6.0.
Centrifuge at ≥ 12,000 x g for 1 minute3. Discard the collection tube with the flow through.
5. Add 400 µl DNA Pre-Wash Buffer to the spin column in a new Collection Tube.
Centrifuge at ≥ 12,000 x g for 1 minute. Empty the collection tube.
6. Add 700 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g for
1 minute. Empty the collection tube.
6
The total yield can be
7. Add 200 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g for improved by eluting the DNA
with 60-70 oC DNA Elution
1 minute. Discard the collection tube with the flow through. Buffer. Also, loading the
eluate a second time,
8. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl4 DNA Elution incubating for 3 minutes at
Buffer or water5 directly on the matrix. Incubate for 5 minutes at room temperature, then room temperature, and
centrifuge at maximum speed for 1 minute to elute the DNA 6. The eluted DNA can be centrifuging again can also
increase total yield.
used immediately for molecular based applications or stored ≤ -20ºC for future use.
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Appendix A
Cell Monolayer Sample Preparation:
The following procedure is designed for up to 5 x 106 monolayer cells. Although cell
types and culture conditions may vary, the protocol will work with high-density growth
cells (e.g., HeLa cells) as well as with low-density growth cells (e.g., neuronal cells).
Trypsinize or scrape adherent cells from a culture flask or plate. Centrifuge the
suspension at approximately 500 x g for 5 minutes. Remove the supernatant and
resuspend the cell pellet in 1 ml PBS (Phosphate Buffered Saline) and then transfer
suspension to a microcentrifuge tube. Centrifuge the suspension at approximately
500 x g for 5 minutes. Discard the supernatant and then follow the Biological Fluids
& Cells workflow on Page 6.
Guidelines for Monolayer Cell DNA Isolation:
Cell numbers (growth densities) can vary between different cell types. Table 1 (below)
provides an approximation of the cell numbers that can be recovered from different
culture containers for “high-density” growth cells like CV1 and HeLa cells.
Table 1: Culture Plate/Flask Growth Area (cm 2) and Cell Number

Culture Container Well /Flask Surface Area Cell Number
96-well plate 0.32-0.6 cm2 4-5 x 104
24-well plate 2 cm2 1-3 x 105
12-well plate 4 cm2 4-5 x 105
6-well plate 9.5 cm2 0.5-1 x 106
T25 Culture Flask 25 cm2 2-3 x 106
T75 Culture Flask 75 cm2 0.6-1 x 107
T175 Culture Flask 175 cm2 2-3 x 107
Buccal Cells and Swabs:
Buccal cells can be isolated using a rinse- or swab-based isolation method.
A. Rinse Method: Vigorously rinse mouth with 10-20 ml of saline solution or

mouthwash orally for 30 seconds. The more vigorous the rinsing action, the more
cells that will be recovered. Spit the saline into a 50 ml tube and pellet the cells at
1,500 rpm for 5 minutes. Discard the supernatant without disturbing the cell
pellet. Continue from Step 1 of the Biological Fluids & Cells workflow on Page 6.
B. Swab Isolation Method: Thoroughly rinse mouth with water before isolating cells.
Brush the inside of the cheek with a buccal swab for 15 seconds (approximately 20
brushes), making sure to cover the entire area of the inner cheek. Rinse the brush
into a microcentrifuge tube using a mixture of 200 µl of BioFluid & Cell Buffer (Red)
and 200 µl DNA Elution Buffer or another isotonic solution. Add 20 µl of Proteinase
K, mix thoroughly, and incubate at 55ºC for 10 minutes. Continue from Step 3 of the
Biological Fluids & Cells workflow on Page 6.
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Appendix B
Samples in DNA/RNA Shield™:
DNA/RNA Shield™ ensures nucleic acid stability during sample storage/transport at

ambient temperatures. There is no need for refrigeration or specialized equipment.
DNA/RNA Shield™ effectively lyses cells and inactivates nucleases and infectious
agents (virus), and it is compatible with various collection and storage devices
(vacutainers, swabs, nasal, buccal, fecal etc.).
DNA/RNA Shield™ purchased separately (R1100 or R1200).
Biological Fluids and Cell Cultures
1. Add 20 µl of Proteinase K to 400 µl of the sample/shield mixture prepared

according to the DNA/RNA Shield™ specifications.
2. Mix thoroughly or vortex 10-15 seconds and then incubate the tube at room
temperature for 20 minutes.
3. Continue from Step 3 of the Biological Fluids & Cells Workflow (pg. 6).
Solid Tissues
1. To each 300 µl sample prepared according the DNA/RNA Shield™ specifications,

add 150 µl Solid Tissue Buffer (Blue) and 10 µl Proteinase K.
2. Mix thoroughly or vortex 10-15 seconds and then incubate the tube at 55ºC 1 – 3
hours.
Note: Overnight digestion at 55°C is possible and will increase the effectiveness of digestion and DNA
recoveries.
3. To remove insoluble debris, centrifuge at ≥ 12,000 x g for 1 minute. Transfer

aqueous supernatant to a clean microcentrifuge tube.
4. Add 1 volume Genomic Binding Buffer to the digested sample. Mix thoroughly
or vortex 10-15 seconds.
5. Continue from Step 4 of the main protocol (pg. 6).
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Notes:
Appendix C
1
If the lysate is still visible
on top of the matrix, Nucleated Blood Samples
centrifuge for another minute
or until completely cleared.
1. Add up to 10 µl of nucleated blood to the following:
BioFluid & Cell Buffer (Red) 200 µl
Proteinase K 20 µl
DNA Elution Buffer (or TE Solution) 200 µl
2. Mix thoroughly by pipetting up and down. Then incubate the tube at 55ºC for
20 minutes.
2
35 µl for highly concentrated Note: The sample may not be completely homogenous before digesting.
3. Add 1 volume of Genomic Binding Buffer to the tube and mix thoroughly by
pipetting up and down and by vortexing. Ensure the sample is homogenous
before continuing.
Note: It may be necessary to pipette up and down many times to ensure the sample is homogenous. Vortexing
will also help ensure the mixture is homogenous.
4. Transfer the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube.

3
DNA Elution Buffer: Centrifuge at ≥ 12,000 x g for 1 minute1. Discard the collection tube with the flow
mM EDTA. If water is used,
through.
make sure the pH is > 6.0.
5. Add 400 µl DNA Pre-Wash to the spin column in a new Collection Tube.
6. Add 700 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g
for 1 minute. Empty the collection tube.
7. Add 200 µl g-DNA Wash Buffer to the spin column directly on the matrix.
4
The total yield can be Centrifuge at ≥ 12,000 x g for 1 minute. Discard the collection tube with the flow
improved by eluting the DNA through.
with 60-70 oC DNA Elution
Buffer. Also, loading the
eluate a second time, 8. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl 2 DNA
incubating for 3 minutes at Elution Buffer or water3 directly on the matrix. Incubate for 5 minutes at room
room temperature, and
centrifuging again can also temperature, then centrifuge at top speed for 1 minute to elute the DNA 4. The
increase total yield. eluted DNA can be used immediately for molecular based applications or stored
≤ -20ºC for future use.
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Notes:
Appendix D
1
Hair and Feather Samples: 35 µl for highly concentrated
1. Freshly prepared DTT (dithiolthreitol) (not provided) needs to be added to the
Proteinase K Digestion and sample (≤ 25 mg) as follows:
Water 90 µl
Solid Tissue Buffer (Blue) 90 µl
DTT (1 M) 10 µl
Proteinase K 10 µl
2. Mix thoroughly or vortex 10-15 seconds and then incubate the tube at 55ºC for 2
DNA Elution Buffer:
1-3 hours. 10 mM Tris-HCl, pH 8.5, 0.1
Note: Overnight digestions are possible without affecting the integrity of the DNA. make sure the pH is > 6.0.
3. Add 400 µl Genomic Binding Buffer to the tube and mix thoroughly by vortexing
for 15 seconds. Centrifuge at ≥ 12,000 x g for 1 minute to pellet insoluble debris.
4. Transfer the mixture (supernatant) to a Zymo-Spin™ IIC-XLR Column in a

Collection Tube. Centrifuge at ≥ 12,000 x g for 1 minute. Discard the collection
tube with the flow through.
3
improved by eluting the DNA
5. Add 400 µl DNA Pre-Wash to the spin column in a new Collection Tube. with 60-70 oC DNA Elution
Centrifuge at ≥ 12,000 x g for 1 minute. Empty the collection tube. Buffer. Alternatively, loading
the eluate a second time,
incubating for 3 minutes at
6. Add 700 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g room temperature, and
for 1 minute. Empty the collection tube. centrifuging again can also
7. Add 200 µl g-DNA Wash Buffer to the spin column directly on the matrix.
Centrifuge at ≥ 12,000 x g for 1 minute. Discard the collection tube with the flow
through.
8. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl 1 DNA

Elution Buffer or water2 directly on the matrix. Incubate for 5 minutes at room
temperature, then centrifuge at top speed for 1 minute to elute the DNA3. The
eluted DNA can be used immediately for molecular based applications or stored
≤ -20ºC for future use.
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Notes: Appendix E
The Quick-DNA™ FFPE Kit FFPE Samples:

(D3067) is specialized for
DNA purification from FFPE
samples.
Deparaffinize FFPE Samples:
1. Remove or trim as much paraffin from the sample(s) as possible (≤ 25 mg).
2. Transfer samples to 1.5 ml microcentrifuge tubes. Add 750 µl xylene (not provided) to the samples.
3. Vortex and incubate samples at room temperature for 1 hour with gentle rocking.
4. Centrifuge at 12,000 x g for 1 minute and remove the xylene from the sample. Repeat steps 2-4.
5. Wash with 1 ml ethanol (100%), vortex briefly, and incubate for 5 minutes with gentle rocking.
Centrifuge at ≥ 12,000 x g for 1 minute, discard the supernatant, and repeat.
6. Wash with 1 ml ethanol (95%), vortex briefly, and incubate for 5 minutes with gentle rocking.
Centrifuge at ≥ 12,000 x g for 1 minute, discard the supernatant, and repeat.
1
It is possible to store 7. Wash with 1 ml ethanol (75%), vortex briefly, and incubate for 5 minutes with gentle rocking.
samples at -80°C at this Centrifuge at ≥ 12,000 x g for 1 minute, discard the supernatant, and repeat.
point for later use.
8. Wash with 1 ml ddiH2O, vortex briefly, and incubate for 5 minutes with gentle rocking. Centrifuge at
≥ 12,000 x g for 1 minute and remove the water from the sample1.
DNA Extraction:
1. Prepare the Proteinase K Digestion to the deparaffinized samples as follows2:
Water 45 µl
Solid Tissue Buffer (Blue) 45 µl
2
If a ≤ 25 mg tissue sample Proteinase K 10 µl
is not fully submerged in the
digestion volume, scale up the 2. Mix thoroughly or vortex 10-15 seconds and incubate the tube at 55 oC for 12-16 hours. Then
digestion to 200 µl while incubate the tube at 94ºC for 20 minutes.
keeping the amount of
Proteinase K the same.
3. Add 6 volumes Genomic Binding Buffer to the tube and mix thoroughly by vortexing for 15
seconds. Centrifuge at ≥ 12,000 x g for 1 minute to pellet insoluble debris.
3
You can elute in as little as 4. Transfer the mixture (supernatant) to a Zymo-Spin™ IIC-XLR Column in a Collection Tube.
35 µl for highly concentrated Centrifuge at ≥ 12,000 x g for 1 minute. Discard the collection tube with the flow through.
5. Add 400 µl DNA Pre-Wash Buffer to the spin column in a new Collection Tube. Centrifuge at ≥
12,000 x g for 1 minute. Empty the collection tube.
4
DNA Elution Buffer: 6. Add 700 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g for 1 minute.
Empty the collection tube.
make sure the pH is > 6.0.
7. Add 200 µl g-DNA Wash Buffer to the spin column directly on the matrix. Centrifuge at ≥ 12,000 x
g for 1 minute. Discard the collection tube with the flow through.
5
improved by eluting the DNA
8. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl 3 DNA Elution Buffer or
with 60-70 oC DNA Elution water4 directly on the matrix. Incubate for 5 minutes at room temperature, then centrifuge at top
Buffer. Also, loading the speed for 1 minute to elute the DNA5. The eluted DNA can be used immediately for molecular
eluate a second time, based applications or stored ≤ -20ºC for future use.
incubating for 3 minutes at
room temperature, and
centrifuging again can also
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Appendix F
Samples Collected onto Storage Papers/Cards:

Rapid purification of inhibitor-free, PCR-quality DNA from blood, saliva, and cells collected
onto Guthrie, FTA®, and other storage papers (cards). Eluted DNA is ideal for PCR,
genotyping, etc.
Additional reagents must be purchased separately. For users who plan to process all 50 or
200 preps with this protocol, please see the following ordering information:
Product Name 50 Preps 200 Preps
1
ZR BashingBead Lysis
ZR BashingBead Lysis Tubes (2.0 mm)1 1 x S6003-50 4 x S6003-50 Tubes (2.0 mm) -
50 pack: D6003-50
BashingBead Buffer2 1 x D6001-3-40 2 x D6001-3-40
3 2
Proteinase K (20 mg) 1 x D3001-2-20 4 x D3001-2-20 BashingBead Buffer -
40 ml: D6001-3-40
4
Solid Tissue Buffer (Blue) 2 x D4068-2-6 3 x D4068-2-22 150 ml: D6001-3-150
5
Genomic Binding Buffer 1 x D4068-3-25 1 x D4068-3-85 3
Proteinase K Set -
5 mg: D3001-2-5
1. Add card samples (punches) to a ZR BashingBead™ Lysis Tube (2.0 mm). Add 400 µl 20 mg: D3001-2-20
BashingBead Buffer to the tube. 4
Solid Tissue Buffer -
6 ml: D4068-2-6
2. Secure lysis tube in a bead beater fitted with a 2 ml tube holder assembly and process at 10 ml: D4068-2-10
maximum speed. 22 ml: D4068-2-22
5
Note: Processing times may be as little as 40 seconds when using high-speed disrupters Genomic Binding Buffer -
(e.g., FastPrep-24, or similar). See manufacturer’s literature for operating instructions. 25 ml: D4068-3-25
45 ml: D4068-3-45
85 ml: D4068-3-85
3. Centrifuge the ZR BashingBead™ Lysis Tube (2.0 mm) at ≥ 10,000 x g for 1 minute.
4. To the lysate in the ZR BashingBead™ Lysis Tube (2.0 mm), add 40 µl Proteinase K
and 360 µl Solid Tissue Buffer (Blue). Mix and then incubate the tube at 55oC for 10-15
minutes.
5. Centrifuge the ZR BashingBead™ Lysis Tube (2.0 mm) at ≥ 10,000 x g for 1 minute.
Transfer 400 µl supernatant to a microcentrifuge tube.
6. Add 800 µl Genomic Binding Buffer to the tube and mix thoroughly.
7. Transfer 600 µl of the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube.

Centrifuge at ≥ 12,000 x g for 1 minute.
6
8. Discard the flow through from the Collection Tube and repeat Step 8. DNA Elution Buffer:
9. Add 400 µl DNA Pre-Wash Buffer to the spin column in a new Collection Tube. make sure the pH is > 6.0.
4
10. Add 700 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g for improved by eluting the
DNA with 60-70 oC DNA
1 minute. Empty the collection tube. Elution Buffer. Also,
loading the eluate a second
11. Add 200 µl g-DNA Wash Buffer to the spin column. Centrifuge at ≥ 12,000 x g for time, incubating for 3
1 minute. Discard the collection tube with the flow through. minutes at room
temperature, and
centrifuging again can also
12. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl6 DNA Elution increase total yield.
Buffer or water directly on the matrix. Incubate for 5 minutes at room temperature, then
centrifuge at maximum speed for 1 minute to elute the DNA. The eluted DNA can be FastPrep is a registered
used immediately for molecular based applications or stored ≤ -20ºC for future use. trademark of Qbiogene, Inc.
ZYMO RESEARCH CORP.

Page 13

Quick Protocol
Biological Fluids & Cells Protocol

Biological Fluids: ≤ 200 µl
Total DNA from whole blood, buffy coat, saliva, sputum, semen, etc. See the Instruction Manual page 2 for special
considerations.
Cultured Cells: ≤ 5x106

Total DNA from E. coli, insect, or mammalian cells (e.g. HeLa cells, buccal cells, HEK-293 cells, etc.). See the Instruction
Manual page 2 for special considerations.
Note: Pellet cells and discard supernatant. Resuspend cell pellets using DNA Elution Buffer or an isotonic buffer (e.g. PBS):
˂ 1 x 106 cells in 100 µl
1-5 x 106 cells in 200 µl
*Add 1,040 µl of Storage Buffer to each 20 mg tube of Proteinase K. Store at -20°C.

1. Add up to 200 µl sample to a microcentrifuge tube and add:
200 µl BioFluid & Cell Buffer (Red)
20 µl Proteinase K
Note: For an input of less than 200 µl Biological Fluid, proportionally decrease BioFluid & Cell Buffer (Red), Proteinase K, and
Genomic Binding Buffer.
2. Mix thoroughly and then incubate the tube at 55ºC for 10 minutes.
3. Add 1 volume Genomic Binding Buffer to the digested sample. Mix thoroughly.
Example: Add 420 µl Genomic Binding Buffer to the 420 µl digested sample.
4. Transfer the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube. Centrifuge (≥ 12,000 x g) for 1
minute. Discard the collection tube with the flow through.
5. Add 400 µl DNA Pre-Wash Buffer to the column in a new Collection Tube and centrifuge for 1 minute. Empty the
Collection Tube.
6. Add 700 µl g-DNA Wash Buffer and centrifuge for 1 minute. Empty the Collection Tube.
7. Add 200 µl g-DNA Wash Buffer and centrifuge for 1 minute. Discard the collection tube with the flow through.
8. To elute the DNA, transfer to a clean microcentrifuge tube. Add ≥ 50 µl DNA Elution Buffer (minimum 35 µl),
incubate for 5 minutes, and then centrifuge for 1 minute.
Ver. 1.2.0
For the full Instruction Manual, visit
http://www.zymoresearch.com/m/D4068
ZYMO RESEARCH CORP.

Page 14

Quick Protocol
Solid Tissues Protocol

Solid Tissues: ≤ 25 mg
Total DNA from tail snips, ear punches, organ biopsies (brain, liver, heart, kidney, muscle, stomach, bladder, intestine,
etc.).
For special sample types including FFPE, hair and feather, see the Instruction Manual page 3.
*Add 1,040 µl of Storage Buffer to each 20 mg tube of Proteinase K. Store at -20°C.
1. To a tissue sample (≤ 25 mg) in a microcentrifuge tube, add a solution of:

95 µl Water
95 µl Solid Tissue Buffer (Blue)
10 µl Proteinase K
2. Mix thoroughly and then incubate the tube at 55ºC for 1-3 hours or until tissue solubilizes. Mix thoroughly.
Note: To remove insoluble debris, pellet by centrifugation at ≥ 12,000 x g for 1 minute. Transfer aqueous supernatant to a clean tube.
3. Add 2 volumes Genomic Binding Buffer to the supernatant. Mix thoroughly.
Example: Add 400 µl Genomic Binding Buffer to the 200 µl supernatant.
4. Transfer the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube. Centrifuge (≥ 12,000 x g) for 1
minute. Discard the collection tube with the flow through.
5. Add 400 µl DNA Pre-Wash Buffer to the column in a new Collection Tube and centrifuge for 1 minute. Empty the
Collection Tube.
6. Add 700 µl g-DNA Wash Buffer and centrifuge for 1 minute. Empty the Collection Tube.
7. Add 200 µl g-DNA Wash Buffer and centrifuge for 1 minute. Discard the collection tube with the flow through.
8. To elute the DNA, transfer to a clean microcentrifuge tube. Add ≥ 50 µl DNA Elution Buffer (minimum 35 µl),
incubate for 5 minutes, and then centrifuge for 1 minute.
For the full Instruction Manual, visit Ver. 1.2.0
http://www.zymoresearch.com/m/D4068
ZYMO RESEARCH CORP.

Page 15
Troubleshooting:
For Technical Assistance, please contact 1-888-882-9682 or E-mail tech@zymoresearch.com.
Problem Possible Causes and Suggested Solutions
Identifying Desired Elution Volume
DNA Elution Guide
The Relationship Between Elution Volume, DNA Yield, and DNA Concentration Using
Porcine Whole Blood. Using a smaller elution volume results in higher concentrations of
DNA samples, but with reduced yields. Using a larger elution volume results in higher DNA
yields, but at a reduced concentration. Choose an elution volume that best fits your
individual application.
Increasing DNA Yields

• The total yield may be improved by eluting the DNA with DNA Elution
Buffer pre-heated to 60-70oC.
• Loading the eluate a second time, incubating for 3 minutes at room

temperature, and centrifuging again can also increase total yield.
DNase Contamination
• Check pipettes, pipette tips, microcentrifuge tubes, etc. for DNase
contamination and exercise the appropriate precautions during the
DNA purification procedure. All reagents and components supplied
with the Quick-DNA™ Miniprep Plus Kit are DNase-free.
DNA Degradation
• If water is used to elute the DNA, ensure that DNase-free water is

used.
• Certain samples are more prone to degradation as a result of the

conditions used for storage and transport (e.g. FFPE Tissue).
ZYMO RESEARCH CORP.

Page 16
Incomplete Debris Removal

• For solid tissue samples, ensure lysate is centrifuged after digestion
to pellet insoluble debris. Ensure pellet is not transferred to the
column.
Incomplete Lysis/Digestion
• Ensure Proteinase K digestions are performed at 55 oC as indicated.
Extend digestion time to 20 minutes if samples are high in protein.
• Vortex samples longer after the addition of Genomic Binding Buffer

to ensure that the lysate is homogenous.
Tissue Input
• For low DNA-containing tissues (e.g. muscle, etc.) using larger
inputs will increase yields (≤ 25 mg).
Low DNA Yield
• If the lysate does not pass through the column or is extremely
viscous, use less input material. Too much tissue can cause cellular
debris to overload the column and result in recovery of dirty DNA.
Elution Procedures
• Ensure the DNA Elution Buffer hydrates the matrix for 5 minutes at
room temperature before centrifugation.
• To increase yields, heat the DNA Elution Buffer to 60-70ºC before

use. You can also load the eluate a second time, incubate at room
temperature for 3 minutes, and centrifuge again.
Procedural Errors
• Ensure the proper digestion buffer is used. See the Purification
Guide on page 5.
• Ensure the correct volume of Genomic Binding Buffer is used. For

plasma and serum samples, use 3 volumes of Genomic Binding
Buffer. See the Purification Guide on page 5 and the Protocol on
page 6.
Procedural Errors
• The column tip is contaminated with wash buffer flow through.
Ensure the tip does not touch the flow through. Empty the collection
tube or use a new collection tube when instructed.
Low DNA Performance • Insufficient centrifugation: Ensure the indicated centrifugation times
and speeds are used. Increase the centrifugation time of the final
wash step by one minute to ensure complete wash buffer removal.
ZYMO RESEARCH CORP.

Page 17
Tissue Input
• Make sure the lysate has passed completely through the matrix
before proceeding to the wash steps.
• If the lysate does not pass through the column or is extremely

viscous, use less input material. Too much tissue can cause cellular
debris to overload the column and leech salts into the DNA eluate.
Low DNA Performance
Continued
RNA in Eluate
• All reagents and components supplied with the Quick-DNA™
Miniprep Plus Kit are designed for RNA removal. Typically if RNA is
in the eluate, too much tissue/sample was used.
• Ensure the proper amount of Genomic Binding Buffer and

corresponding digestion buffer is used. See the Purification Guide on
page 5.
• Ensure Proteinase K digestions are performed at 55oC as indicated.
• For applications sensitive to trace amounts of RNA, additional RNA

removal may be necessary using an RNase A treatment.
ZYMO RESEARCH CORP.

Page 18
Ordering Information
Product Description Catalog No. Kit Size
D4068 50 preps.
D4069 200 preps.
Quick-DNA™ Microprep Plus Kit D4074 50 preps.
D4070 2 x 96 preps.
Quick-DNA™ 96 Plus Kit
D4071 4 x 96 preps.
For Individual Sale Catalog No. Amount
D3001-2-5 5 mg set
Proteinase K & Storage Buffer
D3001-2-20 20 mg set
D4068-1-2.5 2.5 ml
BioFluid & Cell Buffer (Red) D4068-1-12 12 ml
D4068-1-45 45 ml
D4068-2-2.5 2.5 ml
Solid Tissue Buffer (Blue) D4068-2-6 6 ml
D4068-2-22 22 ml
D4068-3-5-S 5 ml
Genomic Binding Buffer D4068-3-25 25 ml
D4068-3-85 85 ml
D3004-5-15 15 ml
DNA Pre-Wash Buffer D3004-5-30 30 ml
D3004-5-50 50 ml
D3004-2-50 50 ml
g-DNA Wash Buffer D3004-2-100 100 ml
D3004-2-200 200 ml
D3004-4-4 4 ml
DNA Elution Buffer D3004-4-10 10 ml
D3004-4-50 50 ml
C1104-25 25 columns
Zymo-Spin™ IIC-XLR Columns
C1104-50 50 columns
C1001-50 50 tubes
Collection Tubes C1001-500 500 tubes
C1001-1000 1,000 tubes
ZYMO RESEARCH CORP.


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INSTRUCTION MANUAL

Quick-DNA™ Miniprep Plus Kit

For Research Use Only Ver. 1.3.1

ZYMO RESEARCH CORP.

Satisfaction of all Zymo

BioFluid & Cell Buffer (Red) 12 ml 45 ml Room Temp.

Zymo-Spin™ IIC-XLR Columns 50 columns 200 columns Room Temp.

ZYMO RESEARCH CORP.

• To isolate cellular and/or cell-free DNA from up to 40 ml of urine samples, see

• Media should be removed before processing by pelleting cells (at approximately

• For mammalian cell samples, it is possible to reduce Proteinase K digestion time

• For samples stored in DNA/RNA Shield™, see Appendix B (pg. 8).

• Media should be removed before processing by pelleting cells (at approximately

ZYMO RESEARCH CORP.

• Overnight Proteinase K digestion at 55˚C is possible (Step 2, pg. 6).

• For hair and feather samples, see Appendix D (pg. 10).

ZYMO RESEARCH CORP.

Zymo Research offers the EZ

DNA Yields Increase Linearly with Increasing

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Quick-DNA™ Miniprep Plus Kit

Biological Fluids & Cells Solid Tissues

BioFluid & Cell Buffer Solid Tissue Buffer

ZYMO RESEARCH CORP.

Cell Monolayer Sample Preparation:

Guidelines for Monolayer Cell DNA Isolation:

Table 1: Culture Plate/Flask Growth Area (cm 2) and Cell Number

Buccal Cells and Swabs:

Buccal cells can be isolated using a rinse- or swab-based isolation method.

A. Rinse Method: Vigorously rinse mouth with 10-20 ml of saline solution or

ZYMO RESEARCH CORP.

Samples in DNA/RNA Shield™:

DNA/RNA Shield™ ensures nucleic acid stability during sample storage/transport at

DNA/RNA Shield™ purchased separately (R1100 or R1200).

Biological Fluids and Cell Cultures

1. Add 20 µl of Proteinase K to 400 µl of the sample/shield mixture prepared

1. To each 300 µl sample prepared according the DNA/RNA Shield™ specifications,

3. To remove insoluble debris, centrifuge at ≥ 12,000 x g for 1 minute. Transfer

5. Continue from Step 4 of the main protocol (pg. 6).

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4. Transfer the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube.

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4. Transfer the mixture (supernatant) to a Zymo-Spin™ IIC-XLR Column in a

8. Transfer the spin column to a clean microcentrifuge tube. Add ≥ 50 µl 1 DNA

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The Quick-DNA™ FFPE Kit FFPE Samples:

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Samples Collected onto Storage Papers/Cards:

7. Transfer 600 µl of the mixture to a Zymo-Spin™ IIC-XLR Column in a Collection Tube.

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Quick-DNA™ Miniprep Plus Kit

Biological Fluids & Cells Protocol

Cultured Cells: ≤ 5x106

*Add 1,040 µl of Storage Buffer to each 20 mg tube of Proteinase K. Store at -20°C.

ZYMO RESEARCH CORP.

Quick-DNA™ Miniprep Plus Kit

Solid Tissues Protocol

*Add 1,040 µl of Storage Buffer to each 20 mg tube of Proteinase K. Store at -20°C.

1. To a tissue sample (≤ 25 mg) in a microcentrifuge tube, add a solution of: