Supporting online information for
A rapid column-based ancient DNA extraction method for increased sample
throughput
Nadin Rohland1,2* , Heike Siedel1 & Michael Hofreiter1,3
1
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103
Leipzig, Germany
2
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
3
Department of Biology, University of York, YO10 5YW, York, UK
*Author for correspondence; e-mail: nrohland@genetics.med.harvard.edu,
fax: (617) 432-7663
Column DNA extraction protocol for degraded hard tissue samples:
Please refer to the initial protocol published (Rohland & Hofreiter, 2007a) or any other
publication about this subject for general considerations and precautions when handling
ancient samples or historical specimen. The most important precaution when handling
such samples is to separate all work on low DNA content samples from high DNA
concentration samples like amplification products, tissue samples or any other material
and equipment that is handled in a regular DNA laboratory; do not exchange equipment
from the “high DNA laboratory” to the “low DNA laboratory” and do not enter the “high
DNA laboratory” before working in the “low DNA laboratory” to prevent contamination.
We also recommend preparing all buffers before handling the samples, because sample
dust from drilling or cutting and grinding can contaminate the buffers.
Although not extensively tested (we usually use 250 or 500mg sample), proportional
down- and up-scaling of the extraction buffer volume and sample amounts can be done.
However, do not use more than 50mg sample powder per ml of extraction buffer. The
binding buffer volume must be adjusted proportionally as described in the procedure to
keep the ratio of extraction to binding buffer and silica as described.
One problem with the amount of silica suspension is that if very low amounts, for
example only 10μl (when using 25mg of sample in 500μl extraction buffer and 250μl
binding buffer) are used, the amount of silica may not be sufficient to bind all DNA
molecules present in the buffer. Therefore, we recommend using at least 50μl of silica
suspension. Another type of problem arises when very large amounts of silica are used,
for example when starting from several grams of sample powder (2g sample powder,
40ml extraction buffer, 20ml binding buffer and 800μl silica suspension). In that case the
silica will, first, not fit into one column, second, the washing steps may be insufficient
and third, the elution volume is not sufficient to get in contact with all silica particles for
DNA release. We recommend using not more that 200μl silica suspension or, if larger
amounts are used, to distribute the silica onto several columns and to increase the total
elution volume.
For convenient handling, we recommend a vacuum manifold for the washing steps, but a
table-top centrifuge can also be used instead. Using a vacuum manifold after the columns
have first been loaded has the advantage that the silica will be evenly distributed over the
surface of the filter and not concentrated on one edge when a centrifuge is used, which
might result in inefficient washing performance. If a centrifuge is used immediately after
application of the silica suspension, the columns can be centrifuged at low speed (3,000g)
for a few seconds, turned by 180 degrees horizontally and spun again at 16,000g for half
a minute to completely remove the binding buffer. If a vacuum manifold is used, we
recommend centrifuging the columns after applying the silica suspension onto the
column for half a minute at 16,000g to remove any remaining binding buffer. Both a
vacuum manifold and a centrifuge are suitable for the following washing steps. In case
the silica is still yellow or brown after the two washing steps recommended, repeating the
washing step several times is usually sufficient to remove any remaining inhibitors. If a
vacuum manifold is used, it is important to centrifuge the columns after the last washing
step for 1 min at 16,000g, to remove the washing buffer as completely as possible before
the elution step.
Here we describe the protocol with the reagents and equipment we are usually using; this
does not imply that there are no other companies, which have identical or similar reagents
and equipment, which are equally well suited and it does not constitute any
recommendation for a specific company.
Reagents, Reagent setup, Equipment
- Extraction buffer
 EDTA disodium salt dihydrate (Sigma, cat. no. E5134)
 Water, HPLC grade (Sigma Aldrich, cat. no. 270733)
 Proteinase K (Sigma, cat. no. P6556)
In case of historical and contemporary samples, we recommend the use of a
non-ionic surfactant and a reducing agent to account for the more intact cell
structure of younger samples.
 (Triton-X 100, [Sigma, cat. no. T8787])
 (DTT, [Sigma, cat. no. 43815])
Prepare extraction buffer with final concentrations of 0.45M EDTA, pH 8.0 and
0.25mg/ml proteinase K, (1% Triton-X 100, 50mM DTT) for the desired amount
of samples.
125 ml of extraction buffer is needed for 24 samples with a maximum of 250 mg
sample powder each (including 5 ml additional buffer).
Always prepare fresh extraction buffer.
- Binding buffer
 Guanidinium thiocyanate (GuSCN) (Sigma, cat. no. G9277)
 Sodium acetate buffer, 3M, pH 5.2 (Sigma, cat. no. S7899)
 Water, HPLC grade (Sigma Aldrich, cat. no. 270733)
Prepare binding buffer with final concentrations of 5M GuSCN and 0.3M sodium
acetate, pH 5.2, for the desired amount of samples.
Approximately 75 ml binding buffer is needed for the binding step and the
application of the silica slurry on the columns for 24 samples.
Do not use binding buffer older than 1 month.
- Silica suspension
 Silicon dioxide, diameter 0.5-10 µm (Sigma Aldrich, cat. no. S5631)
 Water, HPLC grade (Sigma Aldrich, cat. no. 270733)
 Hydrochloric Acid, 30% (Sigma Aldrich, cat. no. 17077)
Prepare a suspension of 4.8 g silicon dioxide in water to a final volume of 40 ml.
After vigorous vortexing, let the large particles settle down for 1 hour. Pipette 39
ml of the supernatant in a new tube and let settle down for additional 4 hours.
Remove and discard 35 ml of the supernatant, that contains the small particles,
and add 48 µl 30% HCl to the 4 ml pellet. Mix carefully, aliquot and store readyto-use silica at room temperature in the dark.
The silica suspension from this preparation should last for 40 samples.
Do not use silica older than 1 month.
Silica particles settle down fast, therefore mix the suspension well by vortexing
immediately before use.
- Washing buffer
 Ethanol absolute (Merk, cat. no. 1.00983.1000)
 Sodium chloride (Sigma Aldrich, cat. no. S7653)
 Water, HPLC grade (Sigma Aldrich, cat. no. 270733)
 EDTA disodium salt dihydrate (Sigma, cat. no. E5134)
 Tris(hydroxymethyl)aminomethane (Tris base) (Sigma, cat no. T1503)
Prepare washing buffer with final concentrations of 50% ethanol, 125mM NaCl,
10 mM Tris and 1 mM EDTA, pH 8.0.
At least 22 ml of washing buffer is needed for 24 samples, but we recommend
preparing more, in case additional washing steps are necessary (if silica is still
colored after 2 washing steps, we recommend more washing steps) and the buffer
can be stored without time limits.
- Column assembly
 MobiCols with 10 µm filter (MobiTec, cat. no. M1002S)
 Filter, glass microfiber binder free Grade GF/B: 1.0 µm (Whatman, cat.
no. 1821-070)
 Hole punch with 7 mm diameter
Cut the desired amount of filters (one per sample) from the microfibre filter using
the 7 mm hole punch and place one filter on top of the large filter (10 µm) already
applied to the column by using the tool that is provided with the MobiCols.
Procedure
- Sample preparation
Equipment
 Drilling or cutting tool with exchangeable drilling bits, cutting discs or
blades. Regular tools, like hammer and chisel, can also be used for large
samples.
 Mortar and pestle or a freezer mill (e.g. SPEX SamplePrep 6750
Freezer/Mill), if pieces of samples need to be ground to a fine powder
Procedure
 Remove the surface of the sample, which you want to cut or where to drill
using a single-use tool. If possible, use a compact part of the bone rather
than spongiosum and use the tooth root rather than enamel.
 Drill into the sample, but use slow speed to prevent overheating, which
could lead to DNA damage, and collect the fine powder.
Alternatively, cut off or out a piece of the sample and grind it to a powder
as fine as possible using mortar and pestle or a freezer mill.
Do not use more than 250 mg per 5 ml.
The finer the powder the better the DNA release during the extraction
step.
- DNA extraction
Reagents
 250mg sample powder per sample
 5ml extraction buffer per sample
 2.5ml binding buffer per sample
 100μl silica suspension per sample
 At least 900μl washing buffer per sample
 50μl 1xTE per sample
Equipment
 Assembled MobiCols, 1 column per sample
 Bench top centrifuge and microcentrifuge
 Rotating wheel or similar equipment, that keeps samples under constant
agitation
 Vacuum manifold and vacuum pump (e.g. QIAvac 24 Plus, Qiagen, cat.
No. 19413; Vacuum Pump, Qiagen, cat. no. 84010) or microcentrifuge
 Disposable VacConnector (Qiagen, cat. no.19407), 1 per sample
 Collection tubes without lids (Qiagen, cat. no. 19201), at least 2 per
sample
Procedure
 Add 5ml extraction buffer to each sample of approximately 250mg sample
powder. Incubate the samples under constant agitation for ~16-24 hours in
the dark at room temperature.
Although not found in our previous tests (Rohland & Hofreiter, 2007b)
DNA yield may increase when incubation is done at 37°C, especially if
younger samples are used.
 Centrifuge the samples for at least 2 min at 5,000g and transfer the
supernatant into a new tube.










Add 2.5ml binding buffer and 100μl silica suspension to the supernatant
and incubate for 3 hours under constant agitation in the dark.
Centrifuge the samples for at least 2 min at 5,000g and discard the
supernatant.
Add 400μl of binding buffer and resuspend the silica pellet by pipetting.
Transfer the silica suspension onto the column, place column on the
vacuum manifold (with a disposable VacConnector between column and
vacuum manifold) and apply vacuum.
Place column in collection tube and centrifuge for 30sec at 16,000g to
remove remaining buffer.
Place column back on the vacuum manifold and add 450μl washing buffer
onto the column, apply vacuum.
Wash at least a second time with 450μl washing buffer. Repeat washing
step if silica is still colored.
Place column in collection tube and centrifuge for 30sec at 16,000g to
remove remaining salts and ethanol.
Place column in fresh 1.5ml tube, add 50μl 1xTE centered on top of the
silica and incubate for 10min at room temperature with closed lids.
Centrifuge for 1min at 16,000g.
(elution step may be repeated; although higher extraction volume will be
obtained, DNA concentration will be lower)
Supporting online information Tables:
Sample ID
233
576
748
751
891
1986
1988
1989
1994
type
bone
bone
tooth
tooth
bone
bone
bone
bone
bone
Origin
Country ID in this study
Potocka zijalka
Slovenia
a
Gailenreuth
Germany
b
Wildkirchli
Switzerland
c
Ossenyaga cave
Russia
d
Gamsulzen cave
Austria
e
Ochsenhalt cave
Austria
f
Ramesch cave
Austria
g
Brieglersberg cave
Austria
h
Schwabenreith cave Austria
i
Table S1: Cave bear samples used in this study. Laboratory internal ID, type of material,
cave in which sample was found (origin), country of origin and ID used in this study.
sample ID
~amount of
sample powder
in mg/ml
50
100
200
400
e
g
i
copy number per gram
(standard deviation)
relative to best method
29,795
9,541
86
(31,319)
(10,436)
(93)
1
4,972
1
3,889
0.22
386
(4,445)
(4,391)
(553)
0.17
1,464
0.41
1,670
1
30
(1,547)
(2,206)
(35)
0.05
0
0
0.18
0
0
0.08
0
0
average
0.74
0.53
0.10 *
0*
Table S2a: Results of qPCR comparing different amounts of sample powder in the
extraction buffer (kept constant at 5ml). Absolute copy numbers (from two measurements
of two dilutions, each) per gram of powder are shown (Standard deviations are given in
parenthesis) together with relative numbers compared to the best performing method for
each sample in this experiment. Significantly worse performing input sample amounts
compared to the best performing ratio are marked with an asterisk (*, P<0.05, paired
Student’s t-test)
sample ID
dilution
~amount of sample
powder in mg/ml
50
1:10
e
1:50
70,570
97,988
79,639
g
1:50
1:250
34,729
0
1:250
1:10
copy number per gram
(standard deviation)
35,530
(8,982)
(8,425)
(34,872)
(5,083)
(15,605)
15,052
15,283
4,043
15,738
21,653
40,857
100
(432)
(2,238)
(5,717)
(3,779)
(11,138)
(13,568)
4,304
4,138
6,383
4,950
6,282
0
(41)
(3,421)
(9,027)
(1,732)
(2,550)
0
0
200
0
0
400
Table S2b: Results of a second qPCR using two of the extracts as in Tab. S2a, but using
further dilution steps comparing different sample powder input and several dilutions. One
extract, i, had too few copies to further dilute it. The copy number for the 1:10 dilution of
the 400mg/ml was not measured, because we found earlier (see Table S2a) that no
amplification occurred at this dilution. Copy number per gram is assessed from two
measurements and standard deviation is given in parenthesis. An inhibitory effect of the
extract due to higher sample input can be excluded, assuming that in this case, copy
number per gram of sample would rise with further dilution as inhibitors in the extract
that would be diluted as well.
Sample ID
ratio of
binding to extraction
buffer
4:1
3:1
2:1
1:1
1:2
e
g
copy number per gram
(standard deviation)
relative to best method
75,459
14,953
(47,356)
(12,876)
0.27
77,986
0.72
15,041
(45,076)
(12,141)
0.28
53,273
0.73
17,905
(30,996)
(14,378)
0.19
18,657
0.87
18,128
(15,016)
(14,880)
0.07
281,309
0.88
20,686
(178,582)
(13,103)
1
1
Average
0.50
0.51
0.53
0.48
1
Table S3a: Results of qPCR comparing different ratios of extraction to binding buffer
(amount of extraction buffer kept constant at 5ml). Absolute copy numbers (from two
measurements of two dilutions, each) per gram of powder are shown together with
relative numbers compared to the best performing method for each sample in this
experiment. All ratios of binding to extraction buffer are not significantly different from
the best performing method after a Student’s t-test (P<0.05).
sample ID
ratio of
binding to extraction
buffer
1:1
1:1.25
1:1.67
1:2.5
1:5
1:10
e
g
copy number per gram
(standard deviation)
relative to best method
20,129
16,887
(20,409)
(18,523)
0.07
58,540
0.75
19,066
(52,342)
(19,377)
0.21
268,207
0.85
18,968
(250,862)
(20,186)
0.94
284,140
0.85
22,437
(287,120)
(16,901)
1
10,757
1
1,452
(11,062)
(1,234)
0.04
26
0.06
886
(52)
(1,482)
0
0.04
average
0.41
0.53
0.90
1
0.05 *
0.02 *
Table S3b: Results of qPCR comparing different ratios of extraction buffer to binding
buffer (amount of extraction buffer kept constant at 5ml) reducing the amount of binding
buffer even further compared to Table S3a. Absolute copy numbers (from two
measurements of two dilutions, standard deviations are given in parenthesis) per gram of
powder are shown together with relative numbers compared to the best performing
method for each sample in this experiment. Significantly worse performing buffer ratios
compared to the best performing ratio are marked with an asterisk (*, P<0.05, Student’s ttest).
sample ID
extraction
method
A
initial
B
columns
a
b
c
d
e
f
copy number per gram
g
h
i
(standard deviation)
relative to best method
8,331
45,498
165
Average
63,209
2,432
1,385
9,724
165
2,027
(43,022)
(2,916)
(725)
(6,613)
(10,974)
(326)
(6,425)
(191)
(1,162)
1
31,091
0.52
4,659
0.6
2,293
0.93
8,957
0.1
467,461
1
22
0.35
28,165
1
74
0.51
3,966
(39,956)
(2,180)
(1,473)
(2,376)
(24,268)
(40)
(5,295)
(113)
(1,399)
0.49
1
1
1
1
0.13
1
0.45
1
0.67
0.79
Table S4: Results of qPCR comparing the initial extraction technique with the new
column-based extraction method on nine cave bear samples. Absolute copy numbers
(from two independent extractions and two measurements of two dilutions, resulting in 8
measurements per sample per method, standard deviations are given in parenthesis) per
gram of powder are shown together with relative numbers compared to the best
performing method for each sample in this experiment. The difference between both
methods is not significant after a Student’s t-test (P<0.05).
success rate
amelogenin
msat loci
location
specimen ID
individual
year found
DNA present
Tai
11782
Lychee*
1992
yes
2/2
18/19
15011
Noah*
2002
yes
2/2
15/19
11791
Xindra*
1992
no
0/2
0/19
15005
Max or Alina
2001
yes
2/2
19/19
11784
Nina
1987
no
0/2
0/19
14992
Gerald
1992
no
0/2
0/19
11796
unknown
1989
yes
2/2
19/19
15007
unknown
unknown
yes
2/2
19/19
11798
unknown
1989
no
0/2
0/19
11797
unknown
1988
no
0/2
0/19
N1254
Gragelli
2005
yes
2/2
19/19
N1253
Mzee
2005
yes
2/2
19/19
N1255
unknown
2005
yes
2/2
19/19
Ngogo
Table S5: Results for the chimpanzee samples. * denote samples that were previously
extracted (Vigilant et al., 2001) and did not yield any DNA then.
Supporting online information References
Rohland N & Hofreiter M (2007a) Ancient DNA extraction from bones and teeth. Nat
Protoc 2, 1756-1762.
Rohland N & Hofreiter M (2007b) Comparison and optimization of ancient DNA
extraction. Biotechniques 42, 343-352.
Vigilant L, Hofreiter M, Siedel H, Boesch C (2001) Paternity and relatedness in wild
chimpanzee communities. Proc Natl Acad Sci U S A 98, 12890-12895.