This protocol may be used to
extract DNA from dry blood spot (DBS) on FTA and other blood card punches with diameters of
approximately 3-6 mm (1/8 – 1/4 inch). It may also be used to extract DNA from
dry blood stains on cloth, papers, swabs, and other fibrous backings of similar
size. The DNA yield is approximately 500 ng per 6-mm punch, which is about 20
times higher than noncommercial extraction methods and 4 times higher than
other commercial kits – a critical factor for increasing detection sensitivity.
1. Place a 6-mm DBS punch (or two 3-mm punches) in a 1.5-ml microfuge tube. Add 200 ul of AquaGenomic solution to the tube.
2. Centrifuge at 10,000-14,000 xg for 1 min to sink the disc into the AquaGenomic solution.
3. Incubate at 70-75 °C for 30 min. After the 30-min incubation,
use a 1-ml pipette tip on a pipette to strike the punch against the
bottom of the tube 10-20 times to smash it and squeeze the solution out
of the matrix (or if available, use a motorized microtube pestle or a
multitube bead beater for processing large number of samples).
.
4. Centrifuge at 10,000-14,000 xg for 5 min to recover the clear
lysate. Transfer the clear lysate (~120 ul) to a new 0.5-ml microfuge
tube. Add 1 vol (~120 ul) of 100% isopropanol and vortex for 60 sec to
mix the contents.
5. Centrifuge at 10,000-14,000 xg for 5-10 min at 22 °C to pellet
the DNA. Decant to discard the supernatant. Carefully fill the tube
with 70% ethanol from a squirt bottle, then flip the tube to discard
the ethanol solution. Be sure to rinse the lid of the tube as well, as
it may catch some reagent. Repeat the 70% ethanol rinse 2 times.
6. Place the tube upside down on a clean paper towel for 5-10 min to
air-dry the DNA pellet. Add 50 ul of TE buffer or deionized water to
the DNA pellet, vortex vigorously to suspend the DNA.
.
AquaGenomic Buccal Swab
Protocol
This
protocol can be used to prepare 10 ug
of genomic DNA from one buccal swab. It is a simple, fast, and
non-invasive method to obtain genomic DNA from individuals.
1.
Harvest the Cells
Swirl and rub
your tongue against the inside of
your cheek and gum for ~5-10 times. Subsequently place a sterile cotton
tipped swab into your mouth. Move the swab around the mouth 10-20 times
to rub different areas along the cheek-gum juncture and soak up the
saliva. After air dry, the swabs can be stored at room temperature for many years.
2.
Extract the DNA
Cut off the swab tip into a 1.5-ml
microfuge tube. Add 200 ul of AquaGenomic Solution to the sample. Incubate at 70-75° C for 15-30 min and then use
a 1-ml tip on a pipette to pound and smack the punch 15-20 times
against the bottom of the tube to squeeze the solution out of the
matrix.
3.
Pellet the Debris
Centrifuge at
14,000 xg for 5 min. Transfer the supernatant (~150 ul) to a new 0.5-ml microfuge tube.
4.
Pellet the DNA
Add 0.8 vol (~120 ul) of 100%
isopropanol and vortex to mix well. Centrifuge at
14,000 xg
for 5 min to pellet the DNA. Decant to discard the supernatant. Fill
the tube
with 70% ethanol from a squirt bottle, then flip the tube to discard
the
ethanol solution. Be sure to rinse the lid of the tube as well, as it
may catch
some reagent. Repeat the 70% ethanol rinse 2 times. Place the tube
upside down
on a clean paper towel for 5-10 min to air-dry the DNA pellet. Add 100 ul
of TE buffer or deionized water to the DNA pellet, pipette or vortex
vigorously
to suspend the DNA.
AquaGenomic Mouthwash
Protocol
Saliva
is one of the most accessible sources of genomic DNA from human
subjects. About 10 ug of genomic DNA can be readily extracted from 200
ul mouthwash using 100 ul of AquaGenomic Solution.
1.
Harvest the Cells
Swirl
and rub your tongue against the inside of your cheek and gum for ~5
times. Spit the saliva into a 50-ml conical tube. Take (do not swallow)
10-20 ml of Scope Mouthwash (or water) and swish it
vigorously around the inside of your mouth for 20 times. Carefully spit
the mouthwash into the tube. Vortex to mix the contents well. Transfer
200 ul mouthwash to a 1.5-ml microfuge tube and centrifuge at
14,000 xg for 1 min to pellet the cells. Aspirate to remove
the supernatant.
2.
Extract the DNA
Add 100 ul of
AquaGenomic Solution to the cell pellet.
Suspend and lyse the cells by vortex vigorously for 60 sec. Incubate at
room
temperature for 15 min (or at 70-75° C for 15 min for better DNA yield).
3.
Pellet the Debris
Vortex
vigorously for 60 sec and centrifuge at
14,000 xg for 5 min to pellet the debris.
4.
Pellet the DNA
Transfer the
supernatant (~90 ul)
to a new 0.5-ml microfuge tube. Add 0.8 vol (~72 ul) of 100%
isopropanol and vortex for 60 sec to mix the contents. Centrifuge at
14,000 xg
for 5 min to pellet the DNA. Decant to discard the supernatant. Fill
the tube
with 70% ethanol from a squirt bottle, then flip the tube to discard
the
ethanol solution. Be sure to rinse the lid of the tube as well, as it
may catch
some reagent. Repeat the 70% ethanol rinse 2 times. Place the tube
upside down
on a clean paper towel for 5-10 min to air-dry the DNA pellet. Add 100 ul
of TE buffer or deionized water to the DNA pellet, pipette or vortex
vigorously
to suspend the DNA.
AquaGenomic
Tail Protocol
Mouse tail is
fibrous, Proteinase K (not supplied) is added to AquaGenomic solution
to assist the disintegration of the tissue. However, if Proteinase K is
not used, overnight incubation is needed. This protocol uses 100 ul of
AquaGenomic solution to prepare 15-20 ug of DNA from
~10 mg (~2 mm) tail snip.
1. Harvest the Tissue
Cut off approximately 2 mm long
fresh or frozen mouse tail. Place the tissue into a microfuge tube
preloaded with 100 ul
of AquaGenomic solution containing 10 ug of Proteinase K (e.g., add 2 ul of 5 mg/ml Proteinase K stock
solution to 100 ul
of AquaGenomic solution just before the extraction).
2. Extract the DNA
Incubate at 65 °C for 2
hours and then at 95 °C for 10 minutes to inactivate the
Proteinase K. The tissue is readily disintegrated by vortexing
vigorously or pipetting.
3. Pellet the Debris
Vortex
vigorously for 60 sec and centrifuge at
14,000 xg for 5 min to pellet the debris. Transfer the supernatant (~90 ul) to a new 0.5-ml microfuge tube.
4. Pellet the DNA
Add 0.8 vol (~72 ul) of 100%
isopropanol and vortex to mix well. Centrifuge at
14,000 xg
for 5 min to pellet the DNA. Decant to discard the supernatant. Fill
the tube
with 70% ethanol from a squirt bottle, then flip the tube to discard
the
ethanol solution. Be sure to rinse the lid of the tube as well, as it
may catch
some reagent. Repeat the 70% ethanol rinse 2 times. Place the tube
upside down
on a clean paper towel for 5-10 min to air-dry the DNA pellet. Add 100 ul
of TE buffer or deionized water to the DNA pellet, pipette or vortex
vigorously
to suspend the DNA. Centrifuge at 14,000 xg for 5 min to pellet any
insoluble
material, and transfer the clear DNA solution to a new tube.
AquaGenomic
Hair Protocol
This
protocol uses 100 ul
of AquaGenomic Solution to prepare 200-400 ng of DNA from 10 hair
follicles.
1.
Harvest the Cells
Pluck
10 individual hairs. Cut the hair follicles (the whitish portion near
the base of the hair) off into a 1.5-ml microfuge tube. To prevent
cross contamination. rinse the scissors with deionized running water,
dip it in ethanol, and then flame it on a Bunsen burner, before use it
to cut the hair follicles of the next subject.
2.
Extract the DNA
Add
100 ul of AquaGenomic Solution to the sample.
Briefly centrifuge to ensure all the hair follicles are submerged in
the solution. Incubate at 70-75°
C for 15 minutes.
3.
Pellet the Debris
Vortex
vigorously for 60 sec and centrifuge at
14,000 xg for 5 min to pellet the debris. Transfer the supernatant (~90 ul) to a new 0.5-ml microfuge tube.
4.
Pellet the DNA
Add 0.8 vol (~72 ul) of 100%
isopropanol and vortex to mix well. Centrifuge at
14,000 xg
for 5 min to pellet the DNA. Decant to discard the supernatant. Fill
the tube
with 70% ethanol from a squirt bottle, then flip the tube to discard
the
ethanol solution. Be sure to rinse the lid of the tube as well, as it
may catch
some reagent. Repeat the 70% ethanol rinse 2 times. Place the tube
upside down
on a clean paper towel for 5-10 min to air-dry the DNA pellet. Add 100 ul
of TE buffer or deionized water to the DNA pellet, pipette or vortex
vigorously
to suspend the DNA.
AquaGenomic
Drosophila Protocol
(Courtesy of Kelly Beumer at The
University of Utah)
Add
50 ul AquaGenomic solution
to 1 fly in 1.5 ml microfugetube.
Homogenize
with Kontes
disposable pestle, either by hand or with the Kontes pellet pestle
motor for 5-10 sec. Up to 48 preps at a time can be done. Leave the
homogenized flies on the desk until all flies have been homogenized.
Incubate
at 70-75˚ C for 20 min.
Vigorously
vortex each tube
20-40 sec.
Centrifuge
sample at max speed
(16000 rpm) for 4 min.
Transfer
supernatant to new tube
containing 1 volume isopropanol. Mix.
Centrifuge
at max speed for 4
min; decant supernatant.
Rinse
with ~500 ul 70% ethanol
by shooting the ethanol solution from a squeeze bottle just below the
rim of the tube. Decant ethanol and pipet off remaining solution.
Allow
tubes to dry ~15 min at
room temperature.
Add
30 ul 10 mM Tris, TE, or ddH2O
to DNA. Leave on bench overnight, vortex, or heat at 60˚ C for one hour
to resuspend.
Store
ateither 4˚ or
–20˚ C. Centrifuge
the DNA solution for 10 min to pellet any insoluble material before use
it for PCR. 0.5
ul of this solution is
sufficient for a PCR reaction.
AquaRNA
Viral Protocol
AquaRNA is suitable for
extracting DNA, RNA, and proteins from double- or single-stranded DNA
viruses and RNA viruses, including bacteriophages. The DNA, RNA, and
protein yields may vary depending on the genome size of the virus and
the viral titer of the starting material but are
close to theoretical values. The starting volume of 50 ml used in this
protocol is for demonstration only; you may use different starting
volume. For example, you may process 1 ml of bacterial culture
containing 109 phage virions with 50 ul of AquaRNA to recover ~2 ng
DNA from phi X174 or ~120 ng DNA from T4 phages,
sufficient for most PCR analysis. You may estimate the viral DNA yield
(or the starting viral volume needed to obtain certain amount of DNA)
using the following equations:
For
double stranded virus: Yield (ng) = Copies (pfu) x Length (bp) x 10-12
For
single stranded virus: Yield (ng) = Copies (pfu) x 0.5 x Length (nt) x
10-12
1. Centrifuge virus
infected culture (50 ml) at 12,000g for 5 min to pellet the cells.
2. Transfer the virus
containing supernatant to a new centrifuge tube (Optional: Add DNase
and RNase to the supernatant and incubate at 37 °C for a few
minutes if complete cellular DNA and RNA removal is needed).
3. Add 1 volume of 20%
PEG8000 in 2.5M NaCl to 5 volumes of virus supernatant (At 3% PEG,
cellular DNA and RNA will not be pelleted with the virions and will be
removed in next step).
4. Vortex to mix and
centrifuge at 12,000g for 10 min to pellet the virions. Decant or
aspirate to remove the PEG supernatant as completely as possible.
5. If the virus is to
be saved as high titer stock, suspend the pellet in 100 ul of 10% glycerol in
PBS and store at -20 °C. Otherwise, add 0.5 ml of AquaRNA
solution to the pellet. Vortex vigorously and incubate at 22 °C
for 15 min to lyse the virions.
6. Transfer the lysate
to a 1.5-ml microfuge tube and add 0.8 volume of isopropanol
to precipitate the viral DNA (or RNA). Vortex to mix well and
centrifuge at 12,000g for 5 min to pellet the DNA (or RNA).
7. If viral proteins
are to be recovered, transfer the protein-containing supernatant to a
new tube for protein precipitation later with 4 volumes of acetone.
Otherwise, decant to discard the supernatant.
8. Rinse the viral DNA
(or RNA) pellet with 75% ethanol 3 times by filling the tube with the
ethanol solution from a squirt bottle and then decanting to discard the
solution. Air-dry the pellet and dissolve the DNA (or RNA) in 50 ul of water or TE buffer.
AquaRNA Small
RNA Protocol
This protocol
can be used to isolate small
RNAs, including 5S RNA, tRNA, microRNA, siRNA, or degraded RNA
from various starting materials. It is essentially the same protocol as
AquaRNA Cell, Tissue, Microbe, and Plant Protocols, except the addition
of a 0.6 volume isopropanol (i.e., 40% final concentration) debris
precipitation step to remove all other cellular components.
1.
Extract the RNA
For bacteria:
Vortex to mix 500 ul of AquaRNA
with 500 ul of
lysozyme-treated bacteria
(from 2.5 ml culture) in a 1.5-ml microfuge tube. Invert the
tube a few times to wet the entire interior of the tube and incubate on
ice for 30 min.
For cell
cultures: Vortex to mix 500 ul of AquaRNA
with 50 ul cell
suspension (~5 million cells) in a
1.5-ml microfuge tube. Invert the tube a few times to wet the entire
interior of the tube and incubate on ice for 30 min.
For tissues:
Homogenize 50 mg of tissue in 500
ul of AquaRNA on ice and incubate on ice for 30 min. Vortex
occasionally. Transfer the homogenate to a 1.5-ml microfuge tube.
2.
Pellet the Debris
Add 0.6 volume
(Do not add more than 0.7 volume
or you will lose some small RNA.) of 100% isopropanol to the above
lysate (for example, add 300 ul isopropanol to 500 ul lysate). Vortex and
invert the tube to mix well.
Centrifuge at 12,000-20,000xg for 10 min to
pellet DNA and large RNA. Transfer the small RNA containing
supernatant to a new microfuge tube.
4. Pellet the Small RNA
Add 0.4 volume
(with respect to the volume of the recovered small RNA containing
supernatant from Step 2 above) of 100% isopropanol to the supernatant
(for example, add 300 ul isopropanol to 750 ul recovered supernatant). Vortex to mix
well. Centrifuge at 12,000-20,000xg for 10 minutes to pellet the small
RNA. Flip to discard the supernatant. Fill the microfuge tube with 75%
ethanol by shooting the ethanol solution from a squeeze bottle at the
cap or sidewall of the tube, and then flip to discard the ethanol.
Repeat the 75% ethanol rinse 2-3 times. Flip to discard residual
ethanol as completely as possible. Air-dry the pellet for ~2-3 minutes.
Add 100 ul of
RNase-free water and vortex to solubilize the RNA. Store the RNA
solution at -20 to -80 °C.
AquaRNA RNase
Decontamination Protocol
AquaRNA may be used to inactivate and
remove residual RNases in purified RNA. Most RNA purification methods
cannot completely remove contaminating RNases from RNA samples. The
purified RNA therefore may be degraded during storage or
analysis. This is the reason that RNA is notoriously known as labile,
unstable, and difficult to work with. To test if your RNA is
contaminated with RNase, you could incubate an aliquot of the sample in
1x DNase I buffer (or any common molecular biology reaction buffer
supplemented with 1 mM CaCl2) at 37 °C for 15 minutes, and then
check the RNA integrity by gel electrophoresis. This RNase
contamination test should be a routine prior to the
start of any RNA experiment. If RNase contamination is
detected, the RNA sample must be de-contaminated with AquaRNA. The
protocol below may be used to inactivate RNases in
100 ul of RNA sample using 100 ul of AquaRNA. You may adjust
the volume of AquaRNA for different sample volumes. (Note: This
protocol may also be used to decontaminate RNases in plasmid DNA prior
to its use in in vitro transcription reactions. It may also be used to
decontaminate DNases in purified DNA samples, reduce protein
contamination, and facilitate long-term storage of DNA samples at room
temperatures.)
1. Add 100 ul of AquaRNA solution to 100 ul of RNA
sample.
2. Vortex to mix well, invert the tube to wet the
entire interior, and incubate the sample
at room temperature for 15 minutes to inactivate
RNases.
3. To recover the RNA, add 200 ul of 90-100%
isopropanol to the sample. Vortex to mix well. Centrifuge at 12,000-20,000 xg for 5 minutes to
pellet the RNA. Decant the tube to discard the supernatant.
4. To rinse the pellet, fill up the tube
with 75% ethanol by gently shooting the ethanol solution from a squirt
bottle at the cap of the tube without disturbing the pellet and decant
to discard the ethanol solution. Repeat the ethanol rinse 2 times.
5. Air dry the pellet and resuspend the
RNA in 100 ul of RNase-free water.
