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1Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Extraction Methodologies
Anthropology/ Biology 3138
Group 3
Presented to Dr. Matheson
Wednesday 9:30-12:30
Apryle Berube(0444381)
___________________________
Teaching Assistant: Franz Masini
__________________________
November 17, 2010
Lakehead University
2Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Introduction
DNA extraction is need to study DNA such as in polymerase chain reaction(PCR) amplification.
The extraction of DNA is comprised of four basic steps which included collection of the cells,
lysing of the cells, removal of protein, and collection of DNA. [2] There are many ways to collect
cells for a sample. One way is to put out a hair with the root intact. Another way is to simply
wipe the inside of a cheek with a cotton swab or swish NaCl in one’s mouth. Basically, all that is
needed in this step is a collection of cells to be studied. There are many reagents used for DNA
extraction, two of which are studied in this lab. The Chelex extraction is a physical extraction
method and the guanadinium thiocyanate/ silica bead method is a chemical extraction. The first
step in DNA extraction is lysing if the cells. Lysing the cells requires a reagent or heat to break
open the cells and release the DNA. There are many ways to do this. In a physical extraction,
such as Chelex, the cells are lysed by heating them in the Chelex solution. This also dentaures
the DNA in a single strand.The guanadinium thiocyanate solution lyses the cells and digests
protein in the sample. [3] Next is the removal of protein. Chelex is a physical extraction and does
not involve purification steps in the extraction, so protein is not removed. For the guanadinium
thiocyanate solution, a wash buffer is added to remove the protein and other cellular debris,
therefore purifying the sample. Collection of DNA in both extraction methods results in the
DNA being suspended in the solution. Certain factors should be considered when selecting an
extracting agent. For example, Chelex is best for the extraction of DNA used in PCR since it
removes contaminating metal ions that catalyze the digestion of DNA.[2] Guanadinium
thiocyanate/ silica bead solution is useful in the extraction of DNA from tissue and hair samples.
[5]
3Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
To further purify samples, sodium acetate is usually added. The sodium ions help to neutralize
the negative charge on the phosphate groups of the DNA backbone thereby precipitating the
DNA. Cold ethanol is then used to precipitate the remaining DNA since it is not soluble in cold
ethanol. The supernatant is removed and what is left is a purified sample.
Once data for DNA sample have been read on a spectrophotometer, the ratio of DNA/protein
must be calculated as well as DNA concentration. Protein in a sample is measured at 280nm and
DNA is measured at 260nm, therefore the ratio of 260nm/280nm equals the amount of DNA in
relation to the amount of protein. This is useful in determining purity of a sample. The ideal ratio
for this is over 1.8 which would indicate approximately 50% protein and 50% DNA in the
sample. The concentration of DNA in a sample is calculated by the formula below:
[DNA]= 50uG/mL x 260nm optical density x dilution factor
To conserve DNA and fill the spectrophotometric cuvettes, the original sample must be diluted
with water. This affects the concentration of the sample hence the reason why the dilution factor
is important to the calculation of DNA concentration. The main reason for the calculation of
concentration of DNA for this experiment is ensure the proper amount of DNA added to
polymerase chain reaction amplification tubes.[1]
Objective
The purpose of this lab is to compare and understand different extraction methods, as well as
observing and quantifying the DNA obtained from a hair/root sample and a cheek cell sample
using spectrophotometric technique
4Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Materials
Hair sample
Scissors
Tweezers
1.5mL eppendorf tubes
50mL conical tubes
500uL Guanidinium thiocyanate solution
-4M Guanadinium thiocyanate
-0.1M Tris-Hydrogenchloride
-0.02M Ethylenediaminetetraacetic acid
-1.3% triton X-100
~500uL Guanidinium thiocyanate solution + silica beads
Mini Vortex(Fisherbrand)
Ice Cooler
Mini Centrifuge(Fisherbrand)
200uL ethanol
500uL of wash buffer
-0.05M Sodium chloride
-50% ethanol
-1mM Ethylenediaminetetraacetic acid
-0.001M Tris-HCl
Sterilized H2O
100-1000 µL Pipette (Thermo Scientific)
20-200 µL Pipette (Thermo Scientific)
20-200 µL Pipette tips
100-1000 µL Pipette tips
Small plastic trash container
5Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Cotton tip swab
500mL Chelex solution
Heat block(Isotemp)
Benchtop centrifuge(Serval Legend Micro)
Thermoscientific Genesys spectrophotometer
1.5mL cuvettes
22.5uL Sodium acetate
560uL cold ethanol
50 ml Conical Tubes
Methods
Chemical Extraction- Guanadiniumthiocyanate solution method
500uL of guanadinuimthiocyanate(GuSCN) solution was pipetted with a 100-1000uL pipette
into a sterile 1.5mL eppendorf tube. The following steps were used to prepare a group control.
The rest of the group also individually prepared 1 sample each, but only one control was needed
for the entire group.
A hair sample was pulled out of the head with a quick, smooth motion to ensure that the root was
intact. The hair was then cut with scissors to a length of approximately 3/8th of an inch being
sure to include the root tip. The hair/root sample was placed in the sample tube with the root
facing down. Both the sample and control were incubated at 56C on the heat block for one hour
under medium agitation.
The samples were then removed from the heat block. The hair sample was removed from the
sample tube and disposed of.
6Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
The tip of a 100-1000uL pipette tip was cut off and approximately 500uL of the
guanadiniumthiocyanate/ silica bead solution was pipetted into the control and sample tubes.
The tubes were then vortexed for 30 seconds and placed on ice for 15 minutes. The tubes were
removed from the ice and vortexed briefly at 5 minute intervals during the 15 minutes on ice.
After the 15 minutes on ice, the tubes were removed and quick-spinned on the mini centrifuge
for 15 seconds.
The supernatant was carefully removed by decanting in a trash container, and then the 20-200uL
pipette was used once there is little supernatant left. Care was taken not to dump or nick the
pellet.
The silica bead pellet was then re-suspended by adding 500uL of wash buffer and vortexing until
re-suspension. This was then quick-spinned on the mini-centrifuge for 15 seconds and the
supernatant was again removed by decanting and then pipetting.
200uL of ethanol were added to both tubes with a 100-1000uL pipette and vortexed for 15
seconds. These were then quick-spinned on the mini-centrifuge for 15 seconds. The supernatant
was again decanted and pipetted out of the tubes. The tubes were then air-dried by setting them
on the heat block until dried.
The pellets were then re-suspended by adding 250uL of sterile water and vortexing. Both the
sample and control were incubated on the heat block for one hour at 56C and stored for the
following lab session.
Physical Extraction- Chelex Method
7Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Using a cotton-tip swab, the inside of the cheek was swabbed for 5 minutes. The cotton-tip swab
was then cut to a length of about 3/8th of an inch and placed inverted in a 1.5mL eppendorf tube.
500uL of Chelex solution was added to the sample tube and a control tube. A clean cotton-tip
swab was cut to a length of 3/8th on an inch and placed in the control tube. The rest of the group
also individually prepared 1 sample each, but only one control was needed for the entire group.
Both tubes were vortexed for 1 minute and incubated on the heat block for 1-2 hours at 56C
under medium agitation. These were then centrifuged on the bench-top centrifuge for 5 minutes
at 12 000 rpm. The cotton-swab tips were removed from the sample and control tubes. Both
tubes were centrifuged for 2 minutes on the bench-top centrifuge. The supernatant from each
tube was then transferred to sterile eppendorf tubes and stored for PCR amplification.
DNA Quantification and Purity
Both the chelex extraction method and guanadinium thiocyanate extraction method tubes were
taken out of storage. From each tube, 10uL of the solution was pipetted with a 2-20uL pipette
into 2 clean eppendorf tubes. These were labeled as un-pure sample and set aside.
The samples were then centrifuged for 1 minute and the guanadinium thiocyanate solution was
vortexed for 30 seconds. Half of each sample was pipetted into sterile eppendorf tubes. The
chelex tube contained 150uL and the guanadinium thiocyanate tube contained 75uL. To this,
10% v/v 3M sodium acetate was added which equals 7.5uL for the GuSCN tube and 15uL for the
chelex tube. These were then vortexed for 1 minute. 2.5X volume of cold ethanol was then
pipetted into each tube - 185uL in the GuSCN tube and 375uL in the chelex tube. These were
placed on ice for 30 minutes. Once this was done, the samples were centrifuged on the bench-top
centrifuge for 5 minutes at 13 000rpm and at 4C. The hinges of the tubes were placed on the
8Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
outside to help find the pellet. The supernatant was then decanted from each tube and a 2-20uL
pipette was used to remove the remaining supernatant. The pellets were air dried on the heat
block set at 37C to speed-up the drying process. Once dry, the pellets were re-suspended by
adding 150uL of sterile water to each and vortexing for 1 minute. Both tubes were then set in the
heat block at 37C for 15 minutes.
Once this was done, the un-pure sample tubes set aside earlier as well as the pure sample tubes
from the previous step were each diluted to 700uL. To the un-pure chelex and GuSCN tubes, a
1:70 dilution was carried out. This means that to 10uL of sample, 690uL of sterile water were
added with a 100-1000uL pipette. The purified samples contained approximately 150uL of
sample, so 100uL of each was pipetted into sterile eppendorf tubes. To each tube, 600uL of
sterile water was added with a 100-1000uL pipette. The remaining 50uL of sample in the chelex
and GuSCN tubes were stored for polymerase chain reaction amplification. Once the dilutions
were carried out, the four samples were pipetted into 1.5mL cuvettes and set in the
spectrophotometer at 260nm. The readings of each were recorded. The spectrophotometer was
then set to 280nm and the readings of the four samples were again recorded.
Results
9Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Table 1 Dilution factor, 260nm and 280nm spectrophotometric readings, 260/280 ratio, and
DNA concentration of Chelex and Guanadinium thiocyanate(GuSCN) pre- and post-purification
Sample Dilution Factor 260nm Reading
280nm Reading
260/280 Ratio
[DNA]ug/mL
Pre-purification GuSCN
1:70(10uL sample:690uL H2O)
0.277 0.213 1.30 0.020
Pre-purification Chelex
1:70(10uL sample:690uL H2O)
0.003 0.004 0.75 0.002
Post-purification GuSCN
1:7(100uL sample:600uL H2O)
0.629 0.621 1.01 4.49
Post-purification Chelex
1:7(100uL sample:600uL H2O)
0.031 0.060 0.52 0.22
Note: See appendix 1.1 for sample calculations for the 260/280 ratio and for [DNA]
Table 2 Pellet and DNA observations for each step of experiment for Chelex, Guanadinium thiocyanate(GuSCN), and control tubes pre- and post-purification
10Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Sample Step Pellet Supernatant Location of DNA Contents of Pellet
Contents of Supernatant or solution
Pre-purification GuSCN
After: incubation
Silica bead Pellet
clear In solution NA Crude extract of proteins and DNA
Quick spin 1 Silica bead Pellet
Clear Pellet DNA crude extract
Proteins and cellular debris
Quick spin 2 Silica bead Pellet
Clear Pellet DNA crude extract
Proteins and cellular debris
Quick spin 3 Silica bead Pellet
Clear Pellet DNA crude extract
Proteins and cellular debris
Air-dry + re-suspension
Silica bead Pellet
A bit cloudy In solution NA DNA crude extact
Chelex After: Incubation
No pellet A bit foamy In solution NA Crude extract of proteins and DNA
Centrifuge 5min
Pellet Clear Supernatant Proteins and other cellular debris
Crude DNA extract
Centriguge 2min
Pellet Clear Supernatant- observation of stringy DNA visible
Proteins and other cellular debris
Crude DNA extract
Chelex Control All Steps performed the same without addition of cheek cells
NONE CLEAR NONe NONE NONE
GuSCN Control All Steps performed the same without addition of hair sample
Pellet of Silica Beads
CLEAR NONE NONE NONE
Post-purification GuSCN
After: Centrifuge
White Pellet Clear Pellet DNA extract
Proteins and cellular debris
Air-dry + re-suspension
NONE Clear In solution NA Purified DNA extract
Chelex After: Centrifuge
White Pellet Clear Pellet DNA Extract
Proteins and cellular debris
Air-dry + re-suspension
NONE Clear In solution NA Purified DNA extract
Discussion
11Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
To extraction methods were studied in this lab. These include the guanidinium
thiocyanate(GuSCN)/silica bead method and the chelex method.
Guanidinium thiocyanate solution(GuSCN) is composed of guanidinium thiocyanate, tris-
hydrogen chloride, ethylenediaminetetraacetic acid, and triton X-100. Ethylenediaminetetraacetic
is used in the sample because of its property of binding metal ions(chelating agent).[10] Triton X-
100 is used to help lyse the cells in a sample and is also useful in protein purification. [9] Tris-HCl
is used as a buffer so that the pH of the solution is within range to prevent DNA degradation. For
the GuSCN/silica bead method as hair sample was used. This contained both the hair shaft and
the root tip. Two types of DNA are present in hair which includes chromosomal DNA and
mitochondrial DNA. Once the hair sample was added to the tube, GuSCN was added to lyse the
cells and dehydrate the DNA since it is a chaotropic agent. GuSCN is also used to denature
protein in the sample. This was also helped by the addition of heat at 56C for 1 hour. After
removal from heat, a solution of GuSCN/ silica beads was added to bind the DNA. Since DNA is
negatively charged, it binds to the positively charged silica beads. This is then placed on ice to
further help the DNA bind and centrifuged to precipitate DNA. A wash buffer composed of tris-
HCl, ethanol, NaCl, and EDTA was then added to remove solubilized proteins, RNA and metal
ions in the sample.[8] The supernatant containing the protein, metal ions, and RNA was then
removed. Ethanol was then added to further purify the sample since DNA is not soluble in
ethanol and therefore precipitates.
The chelex extraction method is a physical extraction and is a simple and cost effective way or
extracting DNA. It is composed of styrene divinlybenzene copolymers with paired
iminodiacetate ions. Because of the harsh environment of chelex, it is not suitable for highly
degraded or low concentrations of DNA. This harsh environment is cause by a pH between 10-
12Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
11 and high temperatures.[7] For this experiment, the inside of the cheek was swabbed to collect
cells. The DNA included in these cells was chromosomal and mitochondrial DNA. Since the
inside of the cheek is filled with bacteria, there might have been a small quantity of pathogenic
and bacterial DNA that would contaminate our sample. Once the tip was added to the Chelex
solution, it was heated for 2 hours at 56C. The heat and chelex caused the cells to lyse in the
sample thereby releasing the DNA. The negatively charged iminodiacetate ions in Chelex
chelate(bind) metal ions out of solution. Mg ions in the sample activate nuclease so removing the
ions prevents this activity.[7] The DNA is located in the supernatant and the resulting precipitate
is composed of metal ions.
An ethanol purification was also performed to further purify the GuSCN sample and to purify the
Chelex sample. Sodium acetate was added to both samples as a source of Na+ for the negatively
charged DNA to bind too. Cold ethanol is then added to precipitate the DNA out of solution.
DNA is insoluble in water because of ethanol’s low dielectric constant. Placing on ice further
improves DNA precipitation because compounds are more soluble at higher temperatures.
Centrifugation helps the suspended precipitate to settle at the bottom.
The main purpose of the extractions is to compare DNA yield. For pre-purification samples, the
GuSCN extraction method yielded a much higher concentration of DNA at 0.020ug/mL than the
Chelex extraction at 0.002ug/mL. This can be seen by the [DNA]ug/mL column readings in
Table 1. This is probably due to the fact that more sample was collected from the hair sample
because it was directly put in the tube compared to the cheek swab which most likely had fewer
cells available. Also, since Chelex is a harsh extraction due to the pH being between 10-11, alot
of the DNA could have been destroyed.[7] Post-purification, the GuSCN still had a higher
concentration of DNA than chelex.
13Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
Purification of the samples had a positive effect on improving DNA concentration. For GuSCN
the concentration rose from 0.020ug/mL to 4.49ug/mL. That is 223.5X increase which is
substantial considering the GuSCN extraction already has a purification step involved. The
chelex sample also increased, but only from 0.002ug/mL to 0.22ug/mL. That accounts for a
110.0X increase. It was expected that the Chelex sample would have a greater increase in yield
compared to the GuSCN sample since it had already been somewhat purified. An explanation for
this might be the fact that there was little DNA to begin with and that a lot of it was accidently
removed during the purification process. The ratios of DNA/protein or 260nm/280nm should
have increased after purification but they have decrease. This would indicate that more DNA
than protein has been lost in the purification process. Common errors could have been dislodging
the pellet and heating to long on the heat block and thereby damaging the DNA. It is expected
that some DNA would be lost in the extraction process, but one would hope that more protein
would be taken out of solution than DNA. The sodium acetate in the purification process not
only causes DNA to precipitate but it also cause proteins to salt-out.[12] Therefore the pellet could
contain a high concentration of protein. Therefore this would explain the lower ratio of 260/280
for the purified samples.
Conclusion
The purpose of the experiment was to compare and understand the two different extraction
methods which were guanidinium thiocyanate/ silica bead and chelex. Much has been learned
about the different methods, but the ratios of DNA post-purification could have been higher.
Purity of the DNA samples is something that should be looked into more to hopefully find better
ways to improve the ratio, however, user technique could also have been a factor that affected
yield.
14Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010
References
[1] “Determination of DNA concentration by Spectrophotometric Estimation.” Accessed Nov.
13, 2010 http://homepages.bw.edu/~mbumbuli/molbio/labs/dna/index.html
[2]Rice, G. DNA Extraction. Montana State University. Accessed Nov.15, 2010
http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html
[3] Capricci, J. “Comparison of two methods of DNA extraction from archaeological bone.”
Lakehead University 1996. pp15-17, 22-23
[4] Matheson, C. Lecture 4.2: Detection of DNA. Lakehead University, Presented Fall 2010
[5]Matheson, C. Lecture 3: Extraction Chemistry. Lakehead University. Presented Fall 2010.
[6] Student Guide to DNA Fingerprinting by PCR. Accessed Nov. 14, 2010
http://www.angelfire.com/mo3/disease/
[7] Chelex Extraction Process. Accessed Nov. 14, 2010
http://www.nfstc.org/pdi/Subject03/pdi_s03_m03_01.htm
[8] Chowdhury, E.D. et al. Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic
detergent. Journal of Biotechnology. Volume 119, Issue 4, 10 October 2005, Pages 343-347
[9] TRITON X-100. Sigma Co.
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/t8532pis.Pa
r.0001.File.tmp/t8532pis.pdf
[10] Ethylenediaminetetraacetic acid. University of Maryland Medical Center
http://www.umm.edu/altmed/articles/ethylenediaminetetraacetic-acid-000302.htm
[11] Mason, P.E. et al. “The hydration structure of guanidinium and thiocyanate ions: Implications for
protein stability in aqueous solution” Stanford University Medical Center. Stanford: October
2001.
[12] Promega. DNA Purification and Quantification. Accessed October 10, 2010
www.promega.com/education/unit004/DNA_Purification_Final.