TECHNICAL MANUAL

Wizard® HMW DNA Extraction Kit

Instructions for Use of Product
A2920

Revised 7/22
TM604
 Wizard® HMW DNA Extraction Kit

All technical literature is available at: www.promega.com/protocols/

Visit the web site to verify that you are using the most current version of this Technical Manual.
E-mail Promega Technical Services if you have questions on use of this system: [email protected]

1. Description.............................................................................................................................................2
2. Product Components and Storage Conditions...............................................................................................3
3. Protocols for HMW Genomic DNA Isolation..................................................................................................3
3.A. Isolating HMW DNA from Whole Blood................................................................................................3
3.B. Isolating HMW DNA from Tissue Culture Cells.....................................................................................5
3.C. Isolating HMW Genomic DNA from Plant Tissue...................................................................................7
3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria......................................................9
4. Troubleshooting.................................................................................................................................... 11
5. References........................................................................................................................................... 12
6. Composition of Buffer............................................................................................................................ 13
7. Related Products .................................................................................................................................. 13
8. Summary of Changes............................................................................................................................. 13

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1
www.promega.com TM604 · Revised 7/22
1. Description
The Wizard® HMW DNA Extraction Kit is designed for isolation of high-molecular-weight (HMW) DNA from white blood
cells (Section 3.A), tissue culture cells (Section 3.B), plant tissue (Section 3.C) and Gram-positive and Gram-negative
bacteria (Section 3.D). Table 1 lists the recommended starting material from each of these sources.
DNA purified with this system is particularly suitable for long-read sequencing platforms when the protocol is closely
followed and DNA handled carefully. Deviating from the recommended protocol, particularly the pipette-mixing or
vortexing instructions, will cause mechanical shearing of HMW DNA and decrease average fragment sizes. Such DNA
shearing will negatively affect performance in long-read sequencing applications. To maximize genomic DNA size, the
protocols for this kit incorporate the use of commonly available 1,000µl wide-bore pipette tips. Using these wide-bore tips
minimizes mechanical damage that may occur during required mixing steps. While these wide-bore tips will yield larger
average fragment sizes, be careful during mixing steps and when handling purified DNA after rehydration. Avoid vigorous
vortexing and use 200µl wide-bore pipette tips to homogenize rehydrated DNA samples, if necessary.
Note: If wide-bore pipette tips are unavailable, you can still extract HMW genomic DNA by substituting an alternate mixing
strategy described in the protocol. This alternative combines the use of standard-bore pipette tips with gentle vortexing to
replace wide-bore pipette mixing. While there is a slight reduction in average size of purified DNA, the yield may increase.

Table 1. Recommendations for Sample Input.

Species and Material Suggested Starting Material

Human Whole Blood 300µl
(Yield varies with
white blood cell count)
Tissue Culture Cells 1–3 × 106 cells
Plant Tissue 40mg
Gram-Negative Bacteria 1ml
Escherichia coli JM109
overnight culture,
~3 × 109 cells/ml
Gram-Positive Bacteria 1ml
Listeria innocua
overnight culture

2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 2. Product Components and Storage Conditions

P R O D U CT SIZE C A T. #

Wizard® HMW DNA Extraction Kit 50 isolations A2920

Each system contains sufficient reagents for 50 miniprep isolations of genomic DNA from a variety of sample types.
Includes:
• 50ml HMW Blood Lysis Buffer
• 25ml HMW Lysis Buffer A
• 1.0ml Proteinase K Solution
• 25ml Protein Precipitation Solution
• 50ml DNA Rehydration Solution
• 250µl RNase A Solution

Storage Conditions: Store the Wizard® HMW DNA Extraction Kit at room temperature (+15°C to +30°C). See product label
for expiration date.

3. Protocols for HMW Genomic DNA Isolation

3.A. Isolating HMW DNA from Whole Blood

We tested the purification of genomic DNA from fresh whole blood collected in EDTA, heparin and citrate anticoagulant
tubes and detected no adverse effects during subsequent DNA manipulations, including PCR (2).
This protocol has been designed and tested for 300µl blood samples. Larger volumes may be processed by first harvesting
the white blood cells, then resuspending and pooling up to three resuspended white blood cell pellets prior to treatment
with HMW Lysis Buffer A. The yield of genomic DNA will vary, depending on the quantity of white blood cells present.
Frozen blood can be used in the following protocols, but yield may be lower than that obtained using fresh blood and
require additional HMW Blood Lysis Buffer.
When handling blood samples, follow the recommended procedures for biohazardous materials at your institution or see
! reference 3.

Materials to Be Supplied by the User

• sterile ClickFit Microtubes, 1.5ml
• standard 1.5ml microcentrifuge tubes
• phosphate-buffered saline (PBS) or TE buffer
• 37°C water bath or dry incubator/shaker
• 56°C water bath or dry incubator/shaker
• isopropanol, room temperature
• 70% ethanol, room temperature
• wide-bore pipette tips (1,000µl and 200µl)
• optional: 65°C water bath (for rapid DNA rehydration)

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 3
www.promega.com TM604 · Revised 7/22
3.A. Isolating HMW DNA from Whole Blood (continued)
1. Add 900µl of HMW Blood Lysis Buffer to a sterile ClickFit Microtube, 1.5ml.

!
Blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.
2. Gently rock the tube of blood until thoroughly mixed; then transfer 300µl of blood to the tube containing the HMW
Blood Lysis Buffer. Invert the tube 5–6 times to mix.
3. Incubate the mixture for 10 minutes at room temperature (invert 2–3 times once during the incubation) to lyse the
red blood cells. Centrifuge at 13,000–16,000 × g for 20 seconds at room temperature.
4. Remove and discard as much supernatant as possible without disturbing the visible white pellet. Approximately
10–20µl of residual liquid may remain in the 1.5ml tube.
5. If blood sample has been frozen, repeat Steps 1–4 until pellet is white. There may be some DNA loss from frozen
samples.
Note: Some red blood cells or cell debris may be visible along with the white blood cells. If the pellet appears bright
red, add an additional aliquot of HMW Blood Lysis Buffer after removing the supernatant above the cell pellet, and
then repeat Steps 3–4.
6. Add 100µl of PBS to the cell pellet and vortex the tube vigorously until the white blood cells are resuspended
(10–15 seconds). If pooling multiple tubes, transfer the 100µl of suspension to the additional tubes in sequence,
vortexing each to suspend the pellets.

!
Completely resuspend the white blood cells to obtain efficient cell lysis.
7. Add 500µl of HMW Lysis Buffer A. Using 1,000µl wide bore pipette tips, mix the solution five times to lyse the white
blood cells. Draw the tube contents slowly from the bottom of the tube, then expel the lysate rapidly down the side
of the tube. The solution should become very viscous. Do not pipet more than five times to avoid DNA shearing.
Notes:
a. If wide-bore pipette tips are unavailable, use standard 1,000µl pipette tips for mixing, gently aspirating and
expelling after adding HMW Lysis Buffer A.
b. If clumps of cells are visible after mixing, incubate the solution at 37°C until the clumps are disappear.
8. Add 3.0µl of RNase A Solution to the lysate and mix the sample by inverting the tube 5–7 times. Incubate the mixture
at 37°C for 15 minutes.
9. Add 20µl of Proteinase K Solution to each lysate and mix the sample by inverting the tube 10 times. Incubate the
mixture at 56°C for 15 minutes. Cool to room temperature for at least 5 minutes or chill on ice 1 minute.
10. Add 200µl of Protein Precipitation Solution to the lysate. Using 1,000µl wide bore pipette tips, aspirate to mix the
solution five times. Draw the tube contents from the bottom of the tube, then expel the lysate rapidly down the side
of the tube. Do not mix by pipette more than five times to avoid DNA shearing. Small protein clumps may be visible
after mixing.
Note: If wide-bore pipette tips are unavailable, vortex for 5 seconds to mix lysate and Protein Precipitation Solution.

4 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 11. Centrifuge at 13,000–16,000 × g for 5 minutes at room temperature. A dark brown protein pellet should be visible. If
any unpelleted debris is visible, repeat the centrifugation step. If no pellet is observed, refer to Section 4,
Troubleshooting.
12. Slowly transfer the supernatant to a clean 1.5ml microcentrifuge by decanting the sample into a tube containing
600µl of room-temperature isopropanol.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in
the tube to avoid contaminating the DNA solution with the precipitated protein.
13. Gently mix the solution by gently inverting the tube eight times. Allow 1 minute at room temperature and repeat the
inversion. White thread-like strands of DNA may become visible. Centrifuge at 13,000–16,000 × g for 2 minutes at
room temperature. The DNA will be visible as a small white pellet.
14. Decant the supernatant and add 600µl of room temperature 70% ethanol to the DNA. Gently invert the tube several
times to wash the DNA pellet and the sides of the microcentrifuge tube. Centrifuge at 13,000–16,000 × g for
2 minutes at room temperature.
15. Carefully aspirate the ethanol. Standard pipette tips can be used for this step. The DNA pellet is very loose at this
point so carefully avoid disturbing or aspirating the pellet into the pipette. Invert the tube on clean absorbent paper
and air-dry the pellet for 10–15 minutes.
16. Add 100µl of DNA Rehydration Solution to the tube. Do not vortex because this will cause mechanical shearing and
decrease average fragment size. Rehydrate the DNA by incubating the solution overnight at room temperature.
Alternatively, incubate the purified DNA at 65°C for 1 hour, periodically mixing the solution by gently tapping the tube.
17. If DNA appears nonhomogeneous (e.g., undissolved pellet is still visible), mix with 200µl wide-bore pipette tips.
Store the DNA at 2–8°C.

3.B. Isolating HMW DNA from Tissue Culture Cells

Materials to Be Supplied by the User

• sterile ClickFit Microtubes, 1.5ml
• standard 1.5ml microcentrifuge tubes
• phosphate-buffered saline (PBS)
• 37°C water bath or dry incubator/shaker
• 56°C water bath or dry incubator/shaker
• isopropanol, room temperature
• 70% ethanol, room temperature
• wide-bore pipette tips (1,000µl and 200µl)
• optional: 65°C water bath, (for rapid DNA rehydration)

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 5
www.promega.com TM604 · Revised 7/22
3.B. Isolating HMW DNA from Tissue Culture Cells (continued)

1. Harvest tissue culture cells, and transfer them to a 1.5ml microcentrifuge tube. For adherent cells, trypsinize the
cells before harvesting.
Note: Do not exceed 3 × 106 cells for each isolation.
2. Centrifuge at 13,000–16,000 × g for 20 seconds to pellet the cells.
3. Remove the supernatant, leaving behind the cell pellet plus 10–50µl of residual liquid.
4. Wash the cells by adding 200µl of PBS and vortexing vigorously to resuspend cells. Centrifuge as instructed in Step 2,
and remove the PBS.
5. Resuspend the pellet in 100µl of PBS by vigorous vortexing.
6. Add 500µl of HMW Lysis Buffer A. Using 1,000µl wide bore pipette tips, mix the solution five times to lyse the cells.
Draw the tube contents slowly from the bottom of the tube, then expel the lysate rapidly down the side of the tube.
The solution should become very viscous. Do not pipet more than five times to avoid DNA shearing.
Notes:
a. If wide-bore pipette tips are unavailable, use standard 1,000µl pipette tips for mixing the HMW Lysis Buffer A.
b. If clumps of cells are visible after mixing, incubate the solution at 37°C until the clumps disappear.
7. Add 3.0µl of RNase A Solution to the nuclear lysate and mix the sample by inverting the tube 5–7 times. Incubate
the mixture at 37°C for 15 minutes.
8. Add 20µl of Proteinase K Solution to each and mix the sample by inverting the tube 10 times. Incubate the mixture at
56°C for 15 minutes. Cool to room temperature for at least 5 minutes or chill on ice for 1 minute.
9. Add 200µl of Protein Precipitation Solution to the nuclear lysate. Using 1,000µl wide bore pipette tips, mix the
solution 5 times. Draw the tube contents from the bottom of the tube, then expel the lysate rapidly down the side of
the tube. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.
Note: If wide-bore pipette tips are unavailable, vortex lysate and Protein Precipitation Solution for 5 seconds. Do
not tip mix.
10. Centrifuge at 13,000–16,000 × g for 10 minutes at room temperature. A whitish protein pellet should be visible. If
any unpelleted debris is visible, repeat the centrifugation step. If no pellet is observed, refer to Section 4,
Troubleshooting.
11. Slowly transfer the supernatant to a clean 1.5ml microcentrifuge by decanting the sample into a tube containing
600µl of room-temperature isopropanol.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in
the tube to avoid contaminating the DNA solution with the precipitated protein.
12. Gently mix the solution by gently inverting the tube eight times. Incubate 1 minute at room temperature and repeat
the inversion. White thread-like strands of DNA may form a visible mass.
13. Centrifuge at 13,000–16,000 × g for 2 minutes at room temperature The DNA will be visible as a small white pellet.

6 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 14. Decant the supernatant and add 600µl of room temperature 70% ethanol to the DNA. Gently invert the tube several
times to wash the DNA pellet and the sides of the microcentrifuge tube. Centrifuge as instructed in Step 13.
15. Carefully aspirate the ethanol. Standard pipette tips can be used for this step. The DNA pellet is very loose at this
point so carefully avoid disturbing or aspirating the pellet into the pipette. Invert the tube on clean absorbent paper
and air-dry the pellet for 10–15 minutes.
16. Add 100µl of DNA Rehydration Solution to the tube. Do not vortex because this will cause mechanical shearing and
decrease average fragment size. Rehydrate the DNA by incubating the solution overnight at room temperature.
Alternatively, incubate the purified DNA at 65°C for 1 hour, periodically mixing the solution by gently tapping the tube.
17. If DNA appears nonhomogeneous (e.g., undissolved pellet is still visible), mix with 200µl wide-bore pipette tips.
Store the DNA at 2–8°C

3.C. Isolating HMW Genomic DNA from Plant Tissue

Materials to Be Supplied by the User

• sterile 1.5ml microcentrifuge tubes
• sterile ClickFit Microtubes, 1.5ml
• liquid nitrogen
• microcentrifuge tube pestle or mortar and pestle
• 65°C water bath
• 37°C water bath
• isopropanol, room temperature
• 70% ethanol, room temperature
• wide-bore pipette tips (1,000µl and 200µl)
1. Process leaf tissue by freezing with liquid nitrogen and grinding into a fine powder using a microcentrifuge tube
pestle or a mortar and pestle. Add 40mg of this leaf powder to a 1.5ml microcentrifuge tube.
2. Add 500µl of HMW Lysis Buffer A, and vortex 1–5 seconds to wet the tissue.
3. Incubate at 65°C for 15–30 minutes.
4. Cool the lysate to room temperature for 5 minutes. Add 3µl of RNase A Solution to the sample and mix by inverting
the tube 5–7 times. Incubate the mixture at 37°C for 15 minutes.
5. Add 20µl of Proteinase K Solution to each leaf sample and mix the sample by inverting the tube 10 times. Incubate
the mixture at 56°C for 15 minutes. Cool to room temperature for at least 5 minutes or chill on ice 1 minute.
6. Centrifuge at 13,000–16,000 × g for 3 minutes at room temperature to pellet any insoluble material. Transfer the
lysate to a clean 1.5ml microcentrifuge tube.
7. Add 200µl of Protein Precipitation Solution to the nuclear lysate. Using 1,000µl wide bore pipette tips, mix the
solution five times. Draw the tube contents from the bottom of the tube, then expel the lysate rapidly down the side
of the tube. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.
Note: If wide-bore pipette tips are unavailable, vortex lysate and Protein Precipitation Solution for 5 seconds. Do
not tip mix.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 7
www.promega.com TM604 · Revised 7/22
3.C. Isolating HMW Genomic DNA from Plant Tissue (continued)
8. Centrifuge at 13,000–16,000 × g for 10 minutes at room temperature. A greenish pellet should be visible. If any
unpelleted debris is visible, repeat the centrifugation step.
9. Slowly transfer the supernatant to a clean 1.5ml microcentrifuge tube by decanting the sample into a tube
containing 600µl of room-temperature isopropanol.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in
the tube to avoid contaminating the DNA solution with the precipitated protein.
10. Gently mix the solution by gently inverting the tube eight times. Incubate for 1 minute at room temperature and
repeat the inversion. White thread-like strands of DNA may form a visible mass.
11. Centrifuge at 13,000–16,000 × g for 2 minutes at room temperature. The DNA may be visible as a small white pellet.
12. Decant the supernatant and add 600µl of room temperature 70% ethanol to the DNA. Gently invert the tube several
times to wash the DNA pellet and the sides of the microcentrifuge tube. Centrifuge as instructed in Step 11.
13. Discard the supernatant then repeat Step 12.
14. Carefully aspirate the ethanol. The DNA pellet is very loose at this point so carefully avoid disturbing or aspirating the
pellet into the pipette. Invert the tube on clean absorbent paper and air-dry the pellet for 10–15 minutes.
15. Add 100µl of DNA Rehydration Solution to the tube. Do not vortex because this will cause mechanical shearing and
decrease average fragment size. Rehydrate the DNA by incubating the solution overnight at room temperature.
Alternatively, incubate the purified DNA at 65°C for 1 hour, periodically mixing the solution by gently tapping the tube.
16. If DNA appears nonhomogeneous (e.g., undissolved pellet is still visible), mix with 200µl wide-bore pipette tips.
Store the DNA at 2–8°C.

8 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria
Purifying genomic DNA from bacteria works best when cultures are grown for 14 hours or less, ideally in exponential
growth phase. Cultures grown for longer tend to produce large amounts of protein and RNA that can affect the
performance and purity of the isolated genomic DNA.

Materials to Be Supplied by the User

• sterile 1.5ml microcentrifuge tubes
• sterile ClickFit Microtubes, 1.5ml
• phosphate-buffered saline (PBS)
• 80°C water bath
• 37°C water bath
• 56°C water bath
• isopropanol, room temperature
• 70% ethanol, room temperature
• wide-bore pipette tips (1,000µl and 200µl)
• 10mg/ml lysozyme (Sigma Cat.# L4919; for Gram-positive bacteria)
• 10mg/ml lysostaphin (Sigma Cat.# L7386; for Gram-positive bacteria)
• optional: 65°C water bath (for rapid DNA rehydration)
1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
2. Centrifuge at 13,000–16,000 × g for 2 minutes to pellet the cells. Remove the supernatant. For Gram-positive bacteria,
proceed to Step 3. For Gram-negative bacteria, resuspend the cells thoroughly in 100µl of PBS, then proceed to Step 5.
3. Add the appropriate lytic enzyme(s) to the cell pellet in a total volume of 100µl, and gently pipet to mix. This treatment
will weaken the cell wall for efficient cell lysis.
Note: For certain Staphylococcus species, a mixture of 60µl of 10mg/ml lysozyme and 60µl of 10mg/ml lysostaphin
is required for efficient lysis. However, many Gram-positive bacterial strains (e.g., Bacillus subtilis, Micrococcus
luteus, Nocardia otitidiscaviarum, Rhodococcus rhodochrous and Brevibacterium albidium) lyse efficiently using
lysozyme alone.
4. Incubate the sample at 37°C for 30–60 minutes.
5. Add 500µl of HMW Lysis Buffer A. Using 1,000µl wide bore pipette tips, mix the solution five times to lyse the cells.
Draw the tube contents slowly from the bottom of the tube, then expel the lysate rapidly down the side of the tube.
The solution should become very viscous. Do not pipet more than five times to avoid DNA shearing.
Note: If wide-bore pipette tips are unavailable, use standard 1,000µl pipette tips for mixing sample with HMW Lysis
Buffer A.
6. If lysis appears incomplete, incubate at 80°C for 5 minutes to lyse the cells then cool to room temperature.
7. Add 3µl of RNase A Solution to the cell lysate, and mix by inverting the tube 5–7 times. Incubate the mixture at 37°C
for 15 minutes.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 9
www.promega.com TM604 · Revised 7/22
3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria (continued)

8. Add 20µl of Proteinase K Solution to each sample and mix by inverting the tube 10 times. Incubate the mixture at
56°C for 15 minutes. Cool to room temperature for at least 5 minutes or chill on ice for 1 minute.
9. Add 200µl of Protein Precipitation Solution to the cell lysate. Using 1,000µl wide bore pipette tips, mix the solution
five times. Draw the tube contents from the bottom of the tube, then expel the lysate rapidly down the side of the
tube. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.
Note: If wide-bore pipette tips are unavailable, vortex lysate and Protein Precipitation Solution for 5 seconds. Do
not tip mix.
10. Centrifuge at 13,000–16,000 × g for 10 minutes at room temperature. A protein pellet should be visible. If any
unpelleted debris is visible, repeat the centrifugation step. If no pellet is observed, refer to Section 4, Troubleshooting.
11. Slowly transfer the supernatant to a clean 1.5ml microcentrifuge tube by decanting the sample into a tube containing
600µl of room-temperature isopropanol.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in
the tube to avoid contaminating the DNA solution with the precipitated protein.
12. Gently mix the solution by gently inverting the tube eight times. Incubate for 1 minute at room temperature and
repeat the inversion. White thread-like strands of DNA may form a visible mass.
13. Centrifuge at 13,000–16,000 × g for 2 minutes at room temperature. The DNA will be visible as a small white pellet.
14. Decant the supernatant and add 600µl of room temperature 70% ethanol to the DNA. Gently invert the tube several
times to wash the DNA pellet and the sides of the microcentrifuge tube. Centrifuge as instructed in Step 13.
15. Carefully aspirate the ethanol. The DNA pellet is very loose at this point so carefully avoid disturbing or aspirating
the pellet into the pipette. Invert the tube on clean absorbent paper and air-dry the pellet for 10–15 minutes.
16. Add 100µl of DNA Rehydration Solution to the tube. Do not vortex because this will cause mechanical shearing and
decrease average fragment size. Rehydrate the DNA by incubating the solution overnight at room temperature.
Alternatively, incubate the purified DNA at 65°C for 1 hour, periodically mixing the solution by gently tapping the tube.
17. If DNA appears nonhomogeneous (e.g., undissolved pellet is still visible), mix with 200µl wide-bore pipette tips.
Store the DNA at 2–8°C.

10 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 4. Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information
available at: www.promega.com. E-mail: [email protected]

Symptoms Causes and Comments

Blood clots present in blood samples The collection tube may have been stored improperly; the blood
was not thoroughly mixed, or inappropriate tubes were used for
drawing blood. Discard the clotted blood and draw new samples
using EDTA-, heparin- or citrate-treated anticoagulant tubes.
Poor DNA yield Blood sample may contain too few white blood cells. Draw new
blood samples.
White blood cell pellet was not resuspended thoroughly in
Section 3.A, Step 5. The white blood cell pellet must be vortexed
vigorously to resuspend the cells.
Blood sample was too old. Best yields are obtained with fresh
blood. Samples that have been stored at 2–8°C for more than
5 days may give reduced yields.
The DNA pellet was lost during isopropanol precipitation. Be
careful when removing the isopropanol to avoid losing the pellet.
Sample is contaminated with RNA. DNA yield can be skewed by
the presence of digested RNA when assayed spectrophotometri-
cally. We recommend the use of DNA-specific fluorometric
assays.
Isolated genomic DNA is not homogeneous, affecting concentra-
tion determination. Purified genomic DNA commonly forms
concentration gradients and can affect the concentration
determination. Mix the sample by flicking the tube several times
or vortex briefly. Do not mix with a pipette. Re-assay the samples.
High RNA contamination Overgrown bacterial culture. When bacterial cultures are grown
longer than 14 hours (stationary phase), they are prone to high
amounts of RNA during purification. Limit growth to exponential
phase.
Poor separation after protein precipitation Added too much sample. Do not exceed the recommended
maximum inputs for sample type. Too much sample results in
excess viscosity, which will affect separations.
Degraded DNA (<50kb in size) Improper collection or storage of the blood sample. Obtain a new
sample under the proper conditions.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 11
www.promega.com TM604 · Revised 7/22
4. Troubleshooting (continued)

Symptoms Causes and Comments

Poor DNA yield using Gram-positive Bacterial culture grown too long. Cultures grown for an
bacteria protocol extended time contain a high proportion of cells that lyse easily
upon exposure to lysostaphin treatment. Start purifications with a
healthy culture.
No protein pellet The sample was not cooled to room temperature before adding
the Protein Precipitation Solution. Cool the sample to room
temperature (at least 5 minutes) or chill on ice for 5 minutes,
vortex for 20 seconds, centrifuge for 3 minutes at
13,000–16,000 × g and proceed with the protocol.
The Protein Precipitation Solution was not thoroughly mixed with
the lysate. Always mix the lysate and Protein Precipitation
Solution completely.
DNA pellet difficult to dissolve Samples may have been overdried. Rehydrate DNA by
incubating for 1 hour at 65°C, and then leave the sample at room
temperature or 4°C overnight. Caution: Do not leave the DNA at
65°C overnight.
Samples were not mixed during the rehydration step. Remember
to mix the samples periodically during the rehydration step. Use
only 200µl wide-bore pipette tips for mixing.

5. References
1. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human
nucleated cells. Nucleic Acids Res. 16, 1215.
2. Beutler, E., Gelbart, T. and Kuhl, W. (1990) Interference of heparin with the polymerase chain reaction.
BioTechniques 9, 166.
3. U.S. Department of Labor, Occupational Safety and Health Administration (1991) Occupational exposure to blood-
borne pathogens, final rule. Federal Register 56, 64175.

12 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM604 · Revised 7/22 www.promega.com
 6. Composition of Buffer

phosphate-buffered saline (PBS) 10X (per liter)

11.5g Na2HPO4
2g KH2PO4
80g NaCl
2g KCl
Dissolve in 1 liter of sterile, deionized water. The pH of 1X PBS will be 7.4.

7. Related Products

DNA Purification Systems

Product Size Cat.#

ProNex Size-Selective Purification System
®
10ml NG2001
ProNex® Size-Selective Purification System 125ml NG2002
ProNex Size-Selective Purification System
®
500ml NG2003
Maxwell® RSC Plant DNA Kit 48 preps AS1490
ReliaPrep™ Blood gDNA Miniprep System 100 preps A5081

8. Summary of Changes
The following changes were made to the 7/22 revision of this document:
1. In Section 3.C, PBS was removed from the Materials to Be Supplied by User list. PBS is not needed in Section 3.C.
2. Miscellaneous text updates were made.
3. Font was updated.

© 2020, 2022 Promega Corporation. All Rights Reserved.

Maxwell, ProNex and Wizard are registered trademarks of Promega Corporation. ReliaPrep is a trademark of Promega Corporation.

Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on
Promega products.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 13
www.promega.com TM604 · Revised 7/22