Low cost and non-toxic genomic DNA extraction for use in molecular marker studies.
Version 1.4, February 28th, 2013. Prepared by Bernhard Hofinger, Owen Huynh and Brad
Till.
1. OBJECTIVE
To develop and adapt a method for low cost and non-toxic genomic DNA extraction for use in molecular
marker studies. We have developed a DNA isolation method based on binding of DNA to silica powder
under chaotropic conditions. The steps of the method involve lysis of plant material, binding of DNA to
silica powder, washing and finally elution of DNA from the silica powder. We have tested the method in
several plant species and show the applicability of such DNA preparations for molecular marker studies
in barley.
2. MATERIALS
MATERIALS FOR LOW-COST DNA EXTRACTIONS
Suppliers and catalogue numbers (where appropriate)
Celite 545 silica powder (Celite 545-AW reagent grade)
Supelco 20199-U
SDS (Sodium dodecyl sulfate) for mol biol approx 99%
Sigma L-4390-250G
Sodium acetate anhydrous
Sigma S-2889 (MW=82.03g/mol)
NaCl (Sodium chloride)
Sigma S-1314-1KG (MW=58.44g/mol)
RNase A
Qiagen RNase A (100 mg/ml)
Ethanol
Ethanol absolut for analysis (Merck 1.00983.2500)
Nuclease-free H2O
Gibco ultrapure distilled water (DNase, RNase-free)
Guanidine thiocyanate
Sigma G9277 (MW=118.2g/mol)
Microcentrifuge tubes (1.5mL, 2.0mL)
Any general laboratory supplier
Micropipettes (1000µL, 200µL, 20µL)
Any general laboratory supplier
Microcentrifuge
Eppendorf Centrifuge 5415D
Optional: Shaker for tubes
Eppendorf Thermomixer comfort for 1.5mL tubes
MATERIALS FOR GRINDING OF LEAF MATERIAL (depending
on grinding method)
Liquid nitrogen
Mortar and pestle, TissueLyser, …
e.g. Qiagen TissueLyser II
Metal beads (tungsten carbide beads, 3mm)
Qiagen Cat.No. 69997
EVALUATION OF DNA YIELD AND QUALITY
DNA concentration
ND-NanoDrop 1000 Spectrophotometer
Agarose gel equipment
Horizontal electrophoresis from any general laboratory
supplier
LOW COST AND NON-TOXIC GENOMIC DNA EXTRACTION FOR USE IN MOLECULAR MARKER STUDIES. VERSION 1.4, PLANT BREEDING AND GENETICS LABORATORY
Page 2
SSR MARKER ANALYSIS
Thermocycler
Biorad C1000 Thermal cycler
PCR tubes
Life Science No 781340
aTaq DNA polymerase (5U/µl)
Promega
PCR buffer without MgCl2, 10x conc
Roche
MgCl2 stock solution (25 mM)
Roche
PCR Nucleotide mix (10 mM)
Promega
Agarose gel equipment
Horizontal electrophoresis from any general laboratory
supplier
SOLUTIONS TO PREPARE:
BUFFER
Receipt
Comments
MW=58.44g/mol
Do not use if percipitate forms. Either
29.22g / 100mL
heat to get fully back into solution or
STOCK SOLUTIONS
5M NaCl stock solution
discard and make fresh
3M Sodium acetate (pH = 5.2)
MW=82.03g/mol
Adjust pH value with glacial acetic acid
24.61g / 100mL
95% (v/v) Ethanol
95 mL ethanol abs + 5 mL H2O
Use fresh. Ethanol absorbs water and
the % will drop over time.
Tris-EDTA (TE) buffer (10x)
Composition:
For 100mL (10x):
Can make Tris and EDTA from
100mM Tris-HCl,
10mL of 1M Tris-
powders. This may be less costsly.
pH=8.0
HCl stock
However, note that the pH of Tris
10 mM EDTA
2mL
of
0.5M
changes with temperature.
EDTA stock
LYSIS BUFFER (standard)
0.5% SDS (w/v) in 10x TE
0.5g SDS /100mL
DNA BINDING BUFFER
6M Guanidine thiocyanate
!!! it takes several hours until dissolved
MW = 118.2 g/mol
(leave it approx. 4-5 hours)
70.92 g / 100mL (6M)
WASH BUFFER
1mL of 5M NaCl + 99mL of 95% EtOH
!!! PREPARE FRESH, because the salt
precipitates during storage
DNA ELUTION BUFFER
depending on application (e.g. TEbuffer; Tris-HCl buffer)
Page 3
3. METHODS (method for centrifuge tubes):
PREPARATION OF SILICA POWDER-DNA BINDING SOLUTION

Fill silica powder (Celite 545 silica) into 50 mL Falcon-tube (to about 2.5mL = approx.
800mg)

Add 30 mL dH2O

Shake vigorously (vortex and invert)

Let slurry settle for approx. 15 min

Remove (pipette off) the liquid

Repeat 2 times (a total of 3 washes)

After last washing step: resuspend the silica powder in about the same amout of water
(up to about 5 mL)

STORE the silica solution at RT until further use (silica : H2O = 1 : 1)
Before use:

suspend stored silica solution (silica : H2O = 1 : 1) by vortexing

Transfer ~50 µL of silica solution to 2mL tubes (prepare 1 tube per sample)
!! try to keep the silica suspended during pipetting to ensure an equal distribution

Add 1mL H2O (a final wash step)

Mix by vortexing

Centrifuge: full speed (13,200) for 10-20 sec

Pipette off liquid

Add 700 µL DNA binding buffer (6M Guanidine thiocyanate)

Suspend the silica powder in DNA binding buffer

The silica binding solution is now ready for further use in the protocol (see Methods)
PREPARATIONS

Prepare 2 mL tubes (1 per sample): add 3 metal beads (tungsten carbide beads) per tube

Harvest leaf material (starting amount of material: about 100 mg fresh weight)
GRINDING
Use appropriate / available grinding protocol (Mortar & pestle, Qiagen TissueLyser)
e.g. grinding using Qiagen TissueLyserII:

Freeze 2mL tubes containing leaf material and 3 metal beads in liquid nitrogen

Grind in TissueLyser by shaking (10 sec at 1/30 speed)

Re-freeze in liquid nitrogen (>30 sec)

Grind again in TissueLyser by shaking (10 sec at 1/30 speed)

Re-freeze in liquid nitrogen (>30 sec)

Keep in liquid nitrogen until lysis buffer is added or store at -80°C for later use
Page 4
LYSIS

Add 800 µL Lysis buffer

Add 4 µL RNaseA (100 mg/ml)

Vortex (~2 min until the powder is dissolved in the buffer)

Incubate: 10min at room temperature

Add 200 µL 3M Sodium Acetate (pH = 5.2)

Mix by inversion of tubes

Incubate on ice for 5 min

Centrifuge 13,200 rpm / 5 min / RT (pellet the leaf material)
DNA BINDING

prepare 700 µL silica binding solution (see above)

transfer 800 µL of the supernatant to the tubes containing silica binding solution)
!! Do not transfer leaf material!

Completely resuspend the silica powder by vortexing and inversion of tubes (ca 20 sec)

incubate 15 min at RT (on a shaker at 400 rpm and/or invert tubes from time to time)

Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)

Remove the supernatant (with pipette)
WASHING (2 times)

Add 500 mL wash buffer (prepared fresh)

Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)

Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)

Repeat the washing step (optional: a third washing step)

Remove the supernatant with pipette (as complete as possible)

optional: short spin and remove residual liquid with pipette

After last washing step: dry the silica in the hood up to 1 hour at RT (make sure there is no wash
buffer left)
RESUSPENSION

Add 200µL TE buffer or 10mM Tris buffer

Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)

Incubate: 20 min / RT / with gentle agitation (on a shaker at 400 rpm and/or invert tubes from time to
time)

Centrifuge (for tubes): 13,200 rpm / 5 min / RT (pellet the silica)

transfer 180 µL supernatant to new tube (avoid transferring silica powder!)

optional: if there is still silica powder in the preps – repeat the centrifugation

check for concentration and integrity of DNA

store the genomic DNA at -20°C for long-term storage or 4°C for short-term storage
Page 5
4. EXAMPLE RESULTS
DNA concentrations and yields using silica powder-based DNA extraction method with different
combinations of buffers
Table 1. Different combinations of self-made buffers and buffers from Qiagen DNeasy Plant Mini kit
Sample
1
2
+
Lysis
DNA binding buffer
DNA wash buffer
-
3
+
-
4
+
-
5
+
-
6
A
B
7
A
B
8
A
B
A
B
Dneasy kit*
Dneasy kit*
Dneasy kit*
Dneasy kit*
Lysis buffer
Lysis buffer
Lysis buffer
Lysis buffer
+Shredder
+Shredder
+Shredder
+Shredder
(our
(our
(our
(our method)
columns
columns
columns
columns
method)
method)
method)
-Shredder
-Shredder
-Shredder
-Shredder
columns
columns
columns
columns
Buffer
Buffer
6M
6M
Buffer
Buffer
6M
6M
AP3/E*
AP3/E*
Guanidine
Guanidine
AP3/E*
AP3/E*
Guanidine
Guanidine
thiocyanate
thiocyanate
thiocyanate
thiocyanate
Buffer AW*
Wash buffer
Buffer AW*
Wash buffer
Buffer AW*
Wash
Buffer AW*
Wash buffer
buffer
DNA concentration
(ng/µL)
14
13
34
41
7
8
4
10
12
11
12
20
10
16
13
17
Total yield (µg)
2.6
2.4
6.2
7.3
1.3
1.5
0.7
1.9
2.2
2.0
2.2
3.5
1.8
2.8
2.4
3.0
*components of Qiagen DNeasy Plant Mini kit
Quality of genomic DNA obtained by silica powder-based DNA extraction method
L
1+
1-
2+
2-
3+
3-
4+
4-
5A
5B
6A
6B
7A
7B
8A
8B
Figure 1. Quality of barley genomic DNA extractions using silica powder and different combinations of self-made
(low-cost) buffers and buffers provided by Qiagen DNeasy kit. 8 µL of each genomic DNA extraction were
separated on a 0.7% agarose gel.
Page 6
1-8: Barley genomic DNA preparation
+: using QIAshredder columns for the preparation of barley leaf lysates (lysis procedure following the kit
instructions)
-: preparation of leaf lysates using the kit instruction (but without using QIAshredder columns
A, B: technical replicates
L: size standard (1 kB Plus DNA ladder - Invitrogen)
All of the genomic DNA preparations show similar DNA concentrations (Table 1) and a good quality of the
genomic DNA on the agarose gel (Figure 1).
Only the DNA preparations “2+” and “2-” (buffer components from the kit in combination with our wash buffer)
show clearly higher concentrations and yields (about 2-3 times higher) than all other DNA preparations. These
results indicate that by modifications of the protocol (i.e. modifications of buffers) some improvements of the DNA
yields are possible.
The DNA preparations of samples 8A and 8B were extracted exclusively with self-made (low-cost) buffers and
show a comparable concentration and yield as the other extractions.
Use of barley genomic DNA obtained by the silica powder-based DNA extraction method for marker
analysis (SSR marker)
The recommended amount of input DNA for SSR analysis are 20ng of DNA. This results in an DNA input volume
between 1uL and 5uL per PCR reaction (e.g. samples “8A” and “8B” : 1.5 and 1.2uL input DNA). We tested
primers for the barley SSR marker cln-130, which amplifies a PCR product of an expected size of about 278 bp.
Primer sequences:
Primer name
Sequence
cnl130_F
AAATGTTGAGCAACGGGAGCT
cnl130_R
ACTTCATAGGGCGGAGGTCT
SSR PCR reactions (Promega aTaq) – per reaction (reaction volume: 25uL):
10x Taq buffer without MgCl2
2.5 µL
MgCl2 (25 mM)
1.5 µL
Nucleotide mix (10 mM)
1.0 µL
Primer forward (10 µM)
0.8 µL
Primer reverse (10 µM)
0.8 µL
aTaq DNA polymerase (5U/µl)
0.25 µL
DNA (20 ng)
1 - 5 µL
H2O (to 25 µL)
17.15 – 13.15 µL
The following PCR program was used (“SSR55mod”):
5 min at 94°C
Page 7
35 cycles (1 min at 94°C; 1 min at 55°C; 30 sec at 72°C)
final extension at 72°C for 5 min
1+
1-
2+
2-
3+
3-
4+
4-
5A
5B 6A 6B
7A
7B
8A 8B
Figure 2. SSR-PCR product amplified from barley genomic DNA extractions (obtained by the use of silica
powder and different combinations of self-made (low-cost) buffers and buffers provided by Qiagen DNeasy
Plant Mini kit – for details see Table1). A 5uL-aliquot of each PCR reaction was checked on a 2.0%
agarose gel.
1-8: Barley genomic DNA preparation
+: using QIAshredder columns for the preparation of barley leaf lysates (lysis procedure
following the kit instructions)
-: preparation of leaf lysates using the kit instruction (but without using QIAshredder columns
A, B: technical replicates
L: size standard (1 kB Plus DNA ladder - Invitrogen)
With the exception of sample 4+, all DNA preparations showed to be suitable for the use in molecular
marker analysis (i.e. SSR). All PCR reactions showed a clear band at the expected size. The very weak
PCR product of sample 4+ corresponds to the low yield of genomic DNA in this specific sample (see
Figure 1 and Table 1).
Application of the silica powder-based DNA isolation method to other plant species
The genomic DNA of maize, rice and Cassava were isolated using the low-cost silica powder-based method with
self-made (low-cost) buffers. 4 technical replicates of each plant species were carried out.
Table 2. DNA concentration and yield of silica powder-based genomic DNA preparations isolated from maize, Cassava and rice.
Page 8
Sample
M1
M2
M3
M4
C1
C2
C3
C4
R1
R2
R3
R4
7.5
6.3
4.7
3.8
37
4.2
13.1
16.1
1.5
1.7
1.1
4.4
1.35
1.13
0.85
0.68
6.66
0.76
2.36
2.9
0.27
0.31
0.2
0.79
DNA
concentration
(ng/µL)
Total yield (µg)
M1-4 (maize 1-4); C1-4 (Cassava 1-4); R1-4 (rice 1-4)
Maize
L
1
2
3
4
Cassava
1
2
3
4
Rice
1
2
3
4
Figure 3. Quality of genomic DNA extractions using silica powder-based DNA isolation method from
maize, Cassava and rice. 8 µL of each genomic DNA extraction were separated on a 0.7% agarose gel.
1-4: Technical replicates of each plant species
L: size standard (1 kB Plus DNA ladder - Invitrogen)
We could successfully apply the silica powder-based extraction method of genomic DNA to several plant
species, i.e. maize, cassava and rice. In all of these extractions, genomic DNA was present, although
some of the sample showed a very low DNA yield (see Table 2 and Figure 3). The cassava DNA
extractions showed the highest yields (up to 37 ng/µL insample “C1”). The maize extractions showed
significantly lower yields: between 3.8 and 7.5 ng/µL. The DNA yields from rice were very low (1.1 – 4.4
ng/µL).
The quality check on the agarose gel revealed some degradation, although the quality should be
sufficient for most standard PCR applications.
5. CONCLUSIONS
Page 9
The silica-powder based low-cost DNA isolation method has been tested with the plant species barley,
maize, rice and cassava. Especially the DNA extractions from barley worked well and provided highquality genomic DNA and sufficient yield. The applicability of the silica-powder based genomic DNA
extractions for molecular marker studies was shown.
The pilot experiments with other plant species (maize, rice and cassava) showed the applicability of the
silica powder DNA extraction method to different economically important plant species. It provided
genomic DNA of suitable quality, but rather low yield. Therefore, some modifications of the protocol to
improve yield and/or DNA quality in different plant species may still be necessary (i.e. rice). Further,
inconsistent recovery from species such as cassava suggests that further optimizations are required.