Jan 04, 2016
96-well DNA Extraction Protocol from 25mm 0.2µm filters
- Virginia Rich1,
- DeLong Lab1
- 1Matthew Sullivan Lab, University of Arizona, Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Virginia Rich, DeLong Lab 2016. 96-well DNA Extraction Protocol from 25mm 0.2µm filters. protocols.io https://dx.doi.org/10.17504/protocols.io.c4hyt5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 18, 2015
Last Modified: February 08, 2018
Protocol Integer ID: 873
Abstract
As used in Rich, Konstantinidis, and DeLong. 2008.
Rich VI., Konstantinidis K., DeLong EF. Design and testing of 'genome-proxy' microarrays to profile marine microbial communities. http://www.ncbi.nlm.nih.gov/pubmed/18028413. Environ. Microbiol., 2008 Feb;10(2):506-21. Epub 2007 Nov 19.
Guidelines
Materials
-2 full ice buckets
-0.2 µm syringe-filters (e.g. Pall Corp. #4192, Acrodisc 25 mm syringe filters, sterile)
-syringes, size depends on volume buffer required; usually 5 ml, 20 ml and 60 ml
-rotating hyb oven set to 37°C
-rotating hyb oven set to 55°C
-later, a heat block or hyb oven (depending on your lysis set up) set to 70°C
-96 well DNA size exclusion columns, (e.g. Edge Biosystems ExcelaPure 96 well UF Plate)
-Qiagen DNeasy 96 Tissue Kit, Qiagen #69581
-1 M Tris, pH 8.3 (kept at my bench, but pH 8.0 - this is OK; e.g. Ambion #9855G)
-0.5 M EDTA (kept at my bench, e.g. Ambion #9260G)
-Sucrose (e.g. stockroom; Mallinckrodt Chemicals #8360-04)
-10% SDS (kept at my bench; e.g. Ambion #9822)
-Lysozyme (kept in door of freezer; e.g. Sigma #L-6876)
-Proteinase K (stored frozen and dry; e.g. EMD #24568-2)
-100 mg/ml RNase (stored at RT, in bench drawer; e.g. Qiagen #1018048)
Protocols relevant to the development of this one:
-The lab's latest 'DNA/RNA Extraction from Large Volume Steripak Filters' protocol, used after the recent Hawaii cruise to extract community DNA for library making.
-The extraction protocol described in Suzuki et al. 2001, the upwelling plume paper; they extracted DNA from 13 mm Supor-200 filters used to filter 30 ml of seawater. They froze filters in 180 µl of a homemade lysis buffer: 20 mM Tris, pH 8; 2 mM EDTA, pH 8, 1.2% TritonX, and 20 mg/ml lysozyme. Once thawed at a later time, they incubated 1 hr at 37°C. Added 1 µl of 15 Kunitz U/ml RNase A, incubated 5' @ RT. Then added 25 µl of a 25 mg/ml ProK (final conc. 3.05 mg/ml). Mixed by vortexing, then followed Qiagen DNeasy Tissue Kit, protocol for Gram-positive bacteria: incubate 1-3 hrs at 55°C, vortex 15 sec, add 200 µl BufferAl (this contains guanidine salt and Tween), incubate 70°C 10', add 200 µl 96-100% EtOH, mix thoroughly by vortexing, and apply to columns.
-Boström et al. 2004 (Ake Hagström's Lab) L&O Methods paper, vol. 2:365-373. Optimization of DNA extraction for quantitative marine bacterioplankton community analysis. Their lysis buffer was 400 mM NaCl, 750 mM sucrose, 20 mM EDTA, 50 mMTris pH 9, and 1 mg/ml final conc. lysozyme. Incubated 30' at 37°C (they also tried 120' but saw worse extraction efficiency, prob due to nuclease activity Tracy thought). Then added SDS to final conc. of 1%, and ProK to final conc. of 100 µg/ml. They incubated at 55°C and saw that an overnight treatment was far better than a shorter treatment. They then proceed with a phenol extraction protocol. Didn't end up using their protocol but mostly because the Steripak one wasn't too diff, and although it only has a 2 hr ProK incubation, it also has a 6.5x higher ProK conc.
-Conversations with Tracy. He typically used 1 mg/ml final lysozyme conc., 200 µg/ml RNase.
- DNeasy 96 Tissue Handbook protocol.
-ExcelaPure 96-well UF PCR Purification protocol.
Expected DNA yield:
Rule of thumb is about 1 fg per genome, 106 genomes per ml, so 1 ng DNA per ml, so 1 µg DNA per L. SO, I filtered 250 mls - 1 L, so I shoud expect (best case scenario) 250 ng - 1 µg DNA out.
Sources by step:
Cell Lysis & RNA Removal: adapted from lab Steripak protocol
Protein Degradation: adapted from lab Steripak protocol
DNA Purification Through DNA-binding Columns: adapted from DNeasy Tissue kit protocol for Gram-positive bacteria, and from DNeasy 96 Tissue kit protocol, and from Marcelino's protocol in the upwelling paper
Final DNA Clean-up & Concentration by Size-Exclusion Columns: from the ExcelaPure 96-well UF PCR purification kit protocol
Purification Through DNA binding Columns Notes:
Principle: selective adsorption of DNA to a silica gel membrane
Notes: According to 96 kit, vol. after steps 1&2 would be about 200 µl, to which they add 410 of buffer AL/E; according to Marcelino's protocol with the old Tissue kit, after steps 1&2 vol. would be 206 µl, to which they added 200 µl buffer AL, incubated 10' at 70, then added 200 µl EtOH. Tech support for 96 kit says to use the slightly higher ratio of buffer to lysate vol that they use in their protocol. They also confirmed that Buffer AL/E, before you add the ethanol, is the same as buffer AL from the old kit.
Note on spins: these are very fast spins, and so balancing is important. If only doing 1 plate, Tracy suggests using a 96 deep well plate as a balance (water squirt bottle). He says it will shake anyway but do not be alarmed. He greased the hinges of the swinging plate holders and said they should be good until next year (2008), and that the plates do fit, barely, in our rig with room to rotate.
The max speed of our rotor is hypothetically 5650 rpm but it will only accept 5250 rpm; 5000 rpm= '4612xg' on the display. So, while the 96 well protocol calls for faster spins, we can't reach them.
Our centrifuge in the main lab does not really recognize the plate spinning rotor; therefore, it does not correctly convert the rpm to the rcf. You can trust the rpm, but not the rcf; that you should calculate yourself.
The equation for interconverting RCF (xg) and RPM is:
RCF=(1.12*radius in mm)*(RPM/1000)2
Thus,Mix our 5000 rpm spins on our rotor, which has a max plate spinning radius of 16 cm but which spins the DNeasy plates at ~12 cm, provides 3360 xg.
Final DNA Clean up & Concentration by Size Exclusion Columns Notes:
Excelapure protocol calls for 100 µl elution, Tracy has gone as low as 20 µl.
Dilute TE is 1:10 regular TE.
Expected DNA yield: for a 250 ml filtration, if only on the order of 105 cells (as in most Hawaii samples, or for sparse MB samples) then only 25 ng of DNA, so 1.25 ng/µl. DNA stores better at higher conc. too.
Materials
MATERIALS
EthanolP212121Catalog #BE-BDH1156
Proteinase KLife TechnologiesCatalog #17916
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
ExcelaPure™ 96-Well UF PCR Purification Plates (No Receivers)Edge BioCatalog #36181
STEP MATERIALS
Proteinase KLife TechnologiesCatalog #17916
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Buffer ALCatalog #19075
EthanolP212121Catalog #BE-BDH1156
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
DEPC-Treated WaterCatalog #4387937
Proteinase KLife TechnologiesCatalog #17916
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Buffer ALCatalog #19075
EthanolP212121Catalog #BE-BDH1156
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
DEPC-Treated WaterCatalog #4387937
Protocol materials
EthanolP212121Catalog #BE-BDH1156
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW1QiagenCatalog #19081
Buffer AEQiagenCatalog #19077
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW2QiagenCatalog #19072
Proteinase KLife TechnologiesCatalog #17916
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
EthanolP212121Catalog #BE-BDH1156
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AEQiagenCatalog #19077
ExcelaPure™ 96-Well UF PCR Purification Plates (No Receivers)Edge BioCatalog #36181
Proteinase KLife TechnologiesCatalog #17916
Buffer ALCatalog #19075
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Buffer ALCatalog #19075
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AW1QiagenCatalog #19081
Buffer AEQiagenCatalog #19077
EthanolP212121Catalog #BE-BDH1156
Buffer AW2QiagenCatalog #19072
DEPC-Treated WaterCatalog #4387937
Proteinase KLife TechnologiesCatalog #17916
DEPC-Treated WaterCatalog #4387937
Proteinase KLife TechnologiesCatalog #17916
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Buffer ALCatalog #19075
EthanolP212121Catalog #BE-BDH1156
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
DEPC-Treated WaterCatalog #4387937
Safety warnings
This protocol is NOT suitable for cleaning up already extracted DNAs! They do not bind effectively to the DNeasy Tissue Columns using this protocol, as written. Set up test plate & vacuum before beginning Final DNA Clean-up & Concentration by Size-Exclusion Columns step.
Prepare lysis buffer:
Note
Make fresh because of sucrose.
Protocol
NAME
Lysis BufferCREATED BY
Bonnie Poulos
Combine EDTA, Tris, and sucrose
| Final Concentration | For 20 ml | For 35 ml | |
| 40 mM EDTA | 1.6 ml of 0.5 M EDTA | 2.8 ml of 0.5 M EDTA | |
| 50 mM Tris (pH 8.3) | 1.0 ml of 1 M Tris (pH 8.3) | 1.75 ml of 1 M Tris | |
| 0.73 M Sucrose | 5.13 g of Sucrose | 8.98 g of Sucrose |
Note
Make fresh because of fructose.
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Shake vigorously to dissolve
Add water to appropriate final volume and shake
Split into two aliquots (15+5 or 30+5)
Note
Can filter-sterilize now but will be sterilizing each aliqout separately so if proceeding immediately (as you should) then no need to double sterilize.
Prepare Lysozyme & RNase Aliquot:
Note
Right before use, add to one aliquot.
Protocol
NAME
Lysozyme & RNase A SolutionCREATED BY
Bonnie Poulos
Combine lysozyme and RNase A
| Final Concentration | For 15 ml | For 30 ml | |
| 1.15 mg/ml Lysozyme | 17.31 mg Lysozyme | 34.62 mg Lysozyme | |
| 200 µg/ml RNase A 100 mg/ml | 30 µl RNase A 100 mg/ml | 60 µl RNase A 100mg/ml |
Note
Note: Using slightly old RNase is fine - I keep mine in a drawer at RT and Chon and others confirmed that several year old RNase should be OK, no need to use extra of it.
Note
Lysozyme should always be prepared fresh on day of use
RNase A catalog # 19101
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Shake to dissolve thoroughly
Filter-sterilize through 0.2 µm filter
Sterile Acrodisc® Syringe Filters with HT Tuffryn® MembraneCatalog #4192
Prepare ProK Aliquot:
Protocol
NAME
Proteinase K SolutionCREATED BY
Bonnie Poulos
Weigh out minimum amount Proteinase K (ProK)
| Final Concentration | For entire 5 ml | For 1.5 ml | For 3.0 ml | |
| 10 mg/ml | 50 mg Proteinase K | 15 mg ProK | 30 mg ProK |
Proteinase KLife TechnologiesCatalog #17916
Add appropriate amount of lysis buffer (from the second 5 ml aliquot)
Note
If you used the full 50 mg of ProK for 5 ml of buffer that would be wasteful since you don't need it. For half-plate you'll need about 1.5 mls to have plenty, for a whole plate you'll need 3 mls.
Shake vigorously to dissolve
Note
It may foam a little.
Filter-sterilize through 0.2 µm syringe filter
Sterile Acrodisc® Syringe Filters with HT Tuffryn® MembraneCatalog #4192
Cell Lysis & RNA removal
Cell Lysis & RNA removal
Thaw filters on ice
Do quick spin down
Transfer each filter to screw top, O-ringed eppendorf tube, also on ice
Note
Note: For my purposes my samples were very precious each time so I didn't muck around, I just used the good, strong, o-ring eppis. However, if one were doing a whole plate's worth of samples or doing this many times, then for lysis you might want to use the rack of collection microtubes in the DNeasy 96 kit, these come with tight caps and are designed for lysis and allow multi-channel pipetting.
Add 250 µl lysis buffer with RNase and lysozyme to each tube
250 µL
Protocol
NAME
Lysis BufferCREATED BY
Bonnie Poulos
Combine EDTA, Tris, and sucrose
| Final Concentration | For 20 ml | For 35 ml | |
| 40 mM EDTA | 1.6 ml of 0.5 M EDTA | 2.8 ml of 0.5 M EDTA | |
| 50 mM Tris (pH 8.3) | 1.0 ml of 1 M Tris (pH 8.3) | 1.75 ml of 1 M Tris | |
| 0.73 M Sucrose | 5.13 g of Sucrose | 8.98 g of Sucrose |
Note
Make fresh because of fructose.
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Shake vigorously to dissolve
Add water to appropriate final volume and shake
Split into two aliquots (15+5 or 30+5)
Note
Can filter-sterilize now but will be sterilizing each aliqout separately so if proceeding immediately (as you should) then no need to double sterilize.
Incubate 37°C for 30 min., rotating end over end at angle, for optimal mixing with minimal frothing
00:30:00
Protein Degradation
Protein Degradation
Add 18.75 µl of Proteinase K solution (10 mg/ml made up in lysis buffer) to a final conc. of 0.65 mg/ml
19 µL
Proteinase KLife TechnologiesCatalog #17916
Add 29.9 µl 10% SDS to a final conc. of 1%
30 µL
SDS, 10% SolutionLife TechnologiesCatalog #AM9822
Incubate at 55°C for 2 hours, rotating end over end at angle
02:00:00
Note
Can also let this step go overnight if needed.
Towards end of this time, turn on heat block or hyb oven to 70°C
Put elution liquid (Buffer AE or water) into 70°C to preheat
DNA Purification Through DNA Binding Columns
DNA Purification Through DNA Binding Columns
Add 300 µl Buffer AL (=Buffer AL/E without the ethanol added)
300 µL
Buffer ALCatalog #19075
Mix thoroughly by vortexing and spin down quickly
Incubate 70°C for 10 mins.
00:10:00
Add 300 µl 96-100% EtOH
300 µL
EthanolP212121Catalog #BE-BDH1156
Mix by vortexing vigorously and spin down quickly
Check pH of lysate, must be <7 to get max. binding efficiency to column
Pipet onto 96 well spin columns, making sure not to whet the rims to avoid cross contamination
Note
Max lysate volume to add to spin columns at one time=900 µl
Place the 96 well column plate onto the S-block for flow through collection
Note
S block should be supplied with kit.
DNeasy 96 Blood & Tissue Kit QiagenCatalog #69581
Seal plate with Airpore tape sheet
Note
Included in kit.
Spin 5788 xg for 10 min. at 40°C
00:10:00
Note
Note: because we can't spin at 40°C, I put the plate set up into a 40°C hyb oven while I prepare the spin balance, so it gets 2-5" at temp before being spun.
Discard flow through
Place in new collection tray
Note
Or in my case just empty out S-block thoroughly and wipe down top rims.
Add additional lysate if needed and repeat spin, etc.
Add 500 µl Buffer AW1, reseal plate
500 µL
Buffer AW1QiagenCatalog #19081
Spin 5788 xg for 5 min. at 40°C
00:05:00
Add 500 µl Buffer AW2, reseal plate
500 µL
Buffer AW2QiagenCatalog #19072
Spin 5788 xg for 5 min. at 40°C
00:05:00
To dry columns, either reseal plate with new sheet and spin 5600 xg for 15 min. at 40°C atop a new collection tray or incubate in hyb oven at 70°C for 15 min.
00:15:00
Transfer column plate to top of rack of "elution microtubes RS"
Add 200 µl pre-heated 70°C Buffer AE or water, reseal plate
200 µL
Note
Note: The only reason to use Ambion water would be if you intend to hyb the crude lysate... which isn't a great idea. You really should do the Final DNA Clean up step for additional clean up, and if you do the Final DNA Clean up step then you can get the DNA in any liquid you want at that stage. SO, you may as well elute here in the Buffer AE since the elution efficiency is highest with that buffer, whose pH is 9.0. If you use water just make sure its pH is >7.0 to permit elution.
Buffer AEQiagenCatalog #19077
Incubate 1 min. at RT
00:01:00
Spin 5788 xg 2 min. to elute
00:02:00
Repeat with second 200 µl (will increase total yield up to 25%) or if you wanted to keep the column small, you could use the first 200 µl elution, heat it back to 70°C, and pass it through the column again (will increase total yield approx. 15%)
Can freeze elutants and break here overnight, or for a while, before proceeding with Final DNA clean up step, particularly if DNA is in Buffer AE which is TE, so will keep the DNA relatively stable
Final DNA Clean up & Concentration by Size Exclusion Columns
Final DNA Clean up & Concentration by Size Exclusion Columns
Transfer the eluted DNA to the 96 well PCR purification plate (no more than 300 µl at a time)
Apply vacuum at 20 inches Hg until dry (~20-30 min.)
00:30:00
Note
Membrane will appear shiny when dry.
Rinse DNA with 100 µl Ambion water, apply vacuum 5-10 min. until dry
100 µL
00:10:00
DEPC-Treated WaterCatalog #4387937
Rinse a second time with water if you want to make sure all Buffer TE removed
Add 20 µl dilute TE
Pipette up and down 20 times and transfer to a clean 96 well plate
Note
Optional: repeat with another 20 µl to ensure all retrieved. DNA stores better at higher concentrations, so elute in minimum possible volume.
Note
PCR plate for temporary or permanent storage, can move to individual tubes for later ease of use.