Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
DNA extraction
DNA is usually extracted from 25 to 30 mg freeze-dried, homogenised (e.g. with a small
coffee grinder) rumen samples. Should you not have access to a freeze-drier, DNA may be
extracted from 250 mg (wet weight) of homogenised rumen sample.
Table 1: DNA extraction methods for the Global Census of Rumen Microbial Diversity Project
Name
Phenol-chloroform plus
bead-beating (PCBB)
methods
Abbreviation
PCSA
PCQI
Repeated bead-beating plus
column method
RBBC
Comments
Phenol-chloroform added
prior to bead-beating step.
Simple and quick protocol
that provides high yields of
DNA.
Modified PCBB, in-house
buffers used, DNA filtered
with QIAquick® PCR
Purification kit, microtitre
plate-adaptable.
Widely used by many
researchers in rumen
microbiology
Reference
Modified from Lueders
et al. 2004. Env
Microbiol 6: 73-78
Kittelmann et al. 2013.
PLoS ONE 8: e47879
Yu and Morrison, 2004.
Biotechniques 36: 808812.
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
Materials and Reagents (molecular biology grade)
The materials and reagents required will depend on the protocol selected for DNA
extraction. Not all reagents and materials are required for each protocol.
Means of mechanical lysis
Mini Beadbeater (Biospec) Fastprep 24 or similar (QBIOgene or MP Biomedicals)
DNA free water
Obtain commercially or prepare as described here. DNA-free water can be prepared by
filtering distilled water through a 0.2-μm pore size sterile filter, autoclaving the water, then
irradiating 2-ml aliquots in 2-ml polypropylene tubes (or for larger volumes use a shallow
container) with UV light (254 nm, 6 W) at a distance of 100 mm for 4 h. Any similar
procedure should suffice.
2 ml screw cap vials suitable for bead beating
Sarstedt Cat. No. 72.693 XXTuff reinforced microvials, Biospec Cat. No. 330TX
0.2 ml PCR tubes
1.5 and 2 ml centrifuge tubes
Zirconia/Silica beads
Diameter 0.1 mm, Biospec Cat. No. 11079101z
Diameter 0.5 mm Biospec Cat. No. 11079105z
Bake these at 180°C for 2 hours prior to use to destroy DNA
120 mM NaPO4 buffer, pH 8
112.87 mM Na2HPO4, 7.12 mM NaH2PO4
Filter-sterilise, autoclave
TNS solution
500 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% SDS (wt/vol), adjust pH with HCl
Filter-sterilise, autoclave
Phenol/Chloroform/Isoamylalcohol (25:24:1)
Chloroform/Isoamylalcohol (24:1)
PEG precipitation solution
30% (wt/vol) polyethylene glycol 6000 in 1.6 M NaCl
Autoclave
70% and 100% ethanol
EB Buffer
10 mM Tris-HCl, pH 8.5
Filter-sterilise, autoclave
SDS 20%
20 g SDS per 100 ml water
Filter-sterilise, autoclave
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
Buffer A
NaCl 200 mM (1.17 g / 100 ml)
Tris 200 mM (2.42 g / 100 ml)
EDTA 20 mM (4 ml of 0.5 M EDTA, pH 8 / 100 ml))
Filter-sterilise, autoclave
Buffer PB
QIAgen Cat. No. 19066 or take from QIAquick PCR purification kit
Buffer PM (microtitre plate format only)
QIAgen Cat. No. 19083 or take from QIAquick PCR purification kit
QIAquick PCR purification kit
QIAgen Cat. No 28104 (50 preps) or 28106 (250 preps)
Lysis Buffer
500 mM NaCl
50 mM Tris-HCl, pH 8.0
50 mM EDTA
4% SDS
10 M ammonium acetate
Isopropanol
TE buffer
10 mM Tris
1 mM EDTA
Filter-sterilize, autoclave
DNAse-free RNAse
10 mg ml-1
Proteinase K, Buffer AL, AW1 and AW2, AE
Take from QIAamp DNA Stool Mini Kit
QIAamp DNA Stool Mini Kit
QIAgen Cat. No. 51504 (50 preps)
3M sodium acetate buffer, pH 5.2
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
Modified phenol-chloroform with bead beating (PCSA method,
modified from Lueders et al., 2004)
1. Add 0.7 g baked 0.1 mm diameter zirconia/silica beads (one 0.2 ml PCR tube) to bead
beating vial with screw cap, autoclave.
2. Add 450 μl 120 mM NaPO4 buffer (pH 8.0).
3. Add 150 μl TNS solution.
4. Add freeze-dried and homogenised rumen sample (25 mg dry weight, 250 mg wet
weight).
5. Extract with 600 μl Phenol/Chloroform/Isoamylalcohol (25:24:1) pH 8.
6. Bead beat vials for 45 s at 6.5 m s-1 (FastPrep 24, QBIOgene or MP Biomedicals) or 4 min at
full speed (Mini Beadbeater, Biospec) and then place them on ice.
7. Centrifuge vials for 20 min at 16,000 × g and 4°C.
8. Take 400 μl supernatant and place in a new 2 ml tube.
9. Extract with 1 vol (400 μl) Chloroform/Isoamylalcohol (24:1), spin 5 min at 16,000 × g and
4°C.
10. Take 350 μl supernatant and mix with 2 volumes PEG precipitation solution (700 μl) and
homogenize by carefully inverting the vial (5 times).
11. Precipitate by spinning for 60 min at 16,000 × g and 4°C.
12. Remove liquid with pipette and add 500 μl cold (4°C) 70 % EtOH to wash pellet.
13. Centrifuge for 10 min at 16,000 × g and 4°C.
14. Optional repeat step 12 and 13 again.
15. Remove as much EtOH as possible carefully by pipetting.
16. Dry DNA under vacuum for 3 min or in a heating block at 50°C for 15 min (or longer, until
dry)
Dried DNA is processed as follows for downstream use
17. Dissolve each DNA precipitate in 200 μl EB buffer (leave to dissolve in the fridge
overnight).
18. Quantify DNA spectrophotometrically or fluorometrically and run on a gel to check for
DNA quality.
19. Aliquot and freeze DNA
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
Modified phenol-chloroform with bead beating and QIAquick PCR
purification kit (tube format – PCQI method)
1. Add 0.7 g baked 0.1 mm diameter zirconia/silica beads (one PCR tube) to bead beating vial
with screw cap, autoclave.
2. Add freeze-dried and homogenised rumen sample (25 mg dry weight, 250 mg wet weight)
to vials containing beads.
3. Add 200 μl of 20% SDS.
4. Add 282 μl Buffer A.
5. Add 268 μl Buffer PB (from QIAquick kit; use Buffer PM if using microtitre plate format).
6. Add 550 μl Phenol/Chloroform/Isoamylalcohol (25:24:1) pH 8 to samples.
7. Bead beat vials for 45 s at 6.5 m s-1 (FastPrep 24, QBIOgene or MP Biomedicals) or 4 min at
full speed (Mini Beadbeater, Biospec) and then place them on ice.
8. Centrifuge samples for 20 min at 16,000 × g and 4°C and then keep on ice.
9. Take the supernatant (500 μl) and transfer into 1.5 ml microcentrifuge tubes.
10. Mix with 650 μl Buffer PB and follow QIAquick PCR purification kit protocol from here.
QIAquick PCR purification kit protocol for rumen samples:
1. Place a QIAquick spin column in a provided 2 ml collection tube.
2. To bind DNA, apply 700 μl sample and Buffer PB mixture to the QIAquick column and
centrifuge for 1 min at 16,000 × g.
a. Note: Column can only accommodate up to 800 μl volume at one time.
3. Discard flow-through. Place the QIAquick column back into the same tube. Repeat Step 2
with the remainder of the sample and Buffer PB mixture.
4. Discard flow-through. Place the QIAquick column back into the same tube.
5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 1 min at 16,000
× g.
6. Discard flow-through and place the QIAquick column back in the same tube.
7. Centrifuge the column for an additional 1 min at 16,000 × g.
a. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the
flow-through is discarded before this additional centrifugation.
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris・HCl, pH 8.5) to the centre of the QIAquick
membrane and centrifuge the column for 1 min at 16,000 × g.
10. Add 30 μl Buffer EB (10 mM Tris・HCl, pH 8.5) to the centre of the QIAquick membrane
and centrifuge the column for 1 min at 16,000 × g to give approximately 80 μl of eluted
DNA.
11. Quantify DNA spectrophotometrically or fluorometrically and run on a gel to check for
DNA quality.
12. Aliquot and freeze DNA
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
RBB+C method (Yu and Morison, 2004)
http://www.biotechniques.com/multimedia/archive/00036/BTN_A_04365ST04_O
_36541a.pdf
I. Cell lysis:
1. Add 0.4 g (0.3 g of 0.1 mm and 0.1 g of 0.5 mm) baked (180°C, 2 h) zirconia/silica beads to
bead beating vial with screw cap, autoclave.
2. Transfer 25 mg of freeze-dried (or 250 mg wet weight) and homogenised sample into the
tube.
3. Add 1 ml of lysis buffer.
4. Homogenize for 4 min (we have extended the original time by one min) at full speed (Mini
Beadbeater, Biospec). Alternatively, 3 min at 6.5 m s-1 (FastPrep 24, QBIOgene or MP
Biomedicals) four steps of 45s each (keep the samples on ice between steps) can also be
used.
5. Incubate at 70°C for 15 min, with gentle shaking by hand every 5 min.
6. Centrifuge at 4°C for 5 min at 16,000 × g. Transfer the supernatant to a fresh 2 ml
microcentrifuge tube, keep the tube on ice.
7. Add 300 μl of fresh lysis buffer to the lysis tube and repeat steps 4-6, and then pool the
supernatant.
II. Precipitation of nucleic acids:
8. Add 260 μl of 10 M ammonium acetate to each lysate tube, mix well, and incubate on
ice for 5 min.
9. Centrifuge at 4°C for 10 min at 16,000 × g.
10. Transfer the supernatant to two 1.5 ml microcentrifuge tubes, add one volume of
isopropanol and mix well, and incubate on ice for 30 min.
11. Centrifuge at 4°C for 15 min at 16,000 × g, remove the supernatant using pipette.
12. Wash the nucleic acids pellet with 1 ml 70% ethanol, at 16000 × g 5 min and 4°C
13. Discard the ethanol and dry the pellet under vacuum for 3 min or in a heating block at
50°C for 15 min (or longer, until dry).
14. Dissolve the nucleic acid pellet in 100 μl of TE (Tris-EDTA) buffer and pool the two
aliquots.
III. Removal of RNA, protein, and purification:
15. Add 2 μl of DNase-free RNase (10 mg/ml) and incubate at 37°C for 15 min.
16. Add 15 μl of proteinase K and 200 μl of Buffer AL (from the QIAamp DNA Stool Mini Kit),
mix well, and incubate at 70°C for 10 min.
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
17. Add 200 μl of ethanol and mix well. Transfer to a QIAamp column and centrifuge at
16,000 × g for 1 min.
18. Discard the flow through, add 500 μl of Buffer AW1 (Qiagen), and centrifuge for 1 min at
room temperature.
19. Discard the flow through, add 500 μl of Buffer AW2 (Qiagen), and centrifuge for 1 min at
room temperature.
20. Dry the column by centrifugation at room temperature for 1 min
21. Add 200 μl of Buffer AE (Qiagen) and incubate at room temperature for 2 min.
22. Centrifuge at room temperature for 1 min at 16,000 × g to elute the DNA.
23. Quantify DNA spectrophotometrically or fluorometrically and run on a gel to check for
DNA quality.
24. Aliquot and freeze DNA
Taken from ‘A Global Census of Rumen Microbial Diversity’, Project Scope and
Protocols, version 5, by Drs Gemma Henderson and Peter Janssen.
Ethanol Precipitation of DNA
Source:
http://kitto.cm.utexas.edu/research/Kittolabpage/Protocols/Microbiology/ethanolPpt.html,
7/7/2010
1. Transfer DNA to a 1.5 ml centrifuge tube
2. Add one tenth volume of cold (4°C) 3M sodium acetate buffer
3. Add at least two volumes of cold (-20°C) 100% ethanol and leave to stand in -20°C freezer
for at least one hour
4. Centrifuge sample for 15 min at highest speed possible in a 4°C micro-centrifuge
5. Remove as much supernatant as possible by pipette, re-centrifuge the pellet at the
highest speed possible for 15 min at 4°C, and then remove as much remaining ethanol as
possible by pipette.
6. Add 200 μL of cold (-20°C) 70% ethanol; centrifuge for 5 minutes at highest speed possible
in a 4 °C centrifuge
7. Remove the supernatant; evaporate the remaining ethanol under vacuum for 3 min or in
a heating block at 50°C for 15 min (or longer, until dry)
8. Re-suspend pellet in desired volume of water or EB buffer