We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.
Note: All reagents used in this protocol are aliquoted from lab stocks into smaller working stocks- this is very important to avoid cross contamination of your DNA samples and contamination of lab stocks. Also, when pipetting, make sure to change pipette tips for every addition to avoid cross contamination.
Part One: Cell Lysis and Digestion
Part Two: DNA Extraction
Part Three: DNA Concentration and Washing
Part Four: Determination of DNA Quality
Part Five: Cesium Chloride Gradient Centrifugation
Representative Results:
When this protocol is done correctly, you should see a gel image after step 4.4 in Determination of DNA quality similar to Figure 1. Actual DNA concentration of extracts will vary depending on the source of the sample. After step 5.7 in CsCl gradient centrifugation, genomic DNA illuminated by blue light should look similar to Figure 2.
Figure 1. 0.8% agarose gel electrophoresis image of high molecular weight DNA collected from four depths in the subarctic Pacific Ocean (in duplicate), stained with the intercalating agent ethidium bromide (10 mg/ml). Gel was run at 15V for ~16hrs in 1X TBE gel running buffer. Sample bands are of good quality showing little evidence of mechanical shearing (shows single bands or smears as opposed to multiple bands) although the 10m extracts retain some RNA carry over (see smear in the 0.5 to 2.0 Kb range).
Figure 2.Genomic DNA band illuminated by blue light after CsCl gradient centrifugation.
Depending on how many samples are to be processed, this can be a time-intensive procedure due to the two one-hour incubation steps, and the repeated washes and centrifugation steps. It is best to plan two whole days for this procedure to leave plenty of time. If there are problems getting the final extract volume in the Amicon tube to reduce down to 200-500μl during DNA concentration and washing, try doing additional TE washes and centrifugations (repeat Part Three) until the appropriate volume is leftover. Also, if the extract smells acutely of chloroform, it is best to do additional washes until the smell lessens to make sure all of the chloroform is removed. Again, all reagents used in this protocol are aliquoted from lab stocks into smaller working stocks- this is very important to avoid cross contamination of your DNA samples and contamination of lab stocks. Also, when pipetting, make sure to change pipette tips for every addition to avoid cross contamination.
We would like to thank the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund and the National Sciences and Engineering Research Council (NSERC) of Canada for supporting ongoing studies on low oxygen regions of coastal and open ocean waters. J.J.W. was supported by fellowships from NSERC and D.A.W. was supported by fellowships from NSERC, Killam and the TULA foundation funded Centre for Microbial Diversity and Evolution. S.L. was supported by the TULA foundation funded Centre for Microbial Diversity and Evolution.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
1 cc syringe (26G5/8 needle) | BD | 309597 | ||
1 ml syringe (26G5/8 needle) | BD | 309597 | ||
1.5ml eppendorf tubes | Eppendorf | 0030 125.150 | ||
15ml tubes | Sigma | CLS430791 | ||
5cc syringe | BD | 309603 | ||
Amicon® Ultra-4 Centrifugal Filter Devices | Millipore | UFC801096 | 10,000 Nominal molecular weight limit | |
Butanol | Fisher | A383-1 | Make it water saturated (butanol:water = 1:1) | |
Centrifuge rotor, JA5.3 | Beckman Coulter | 368690 | ||
Centrifuge, Avanti® J-E | Beckman Coulter | 369003 | ||
Cesium chloride | Sigma | C4036 | ||
Chloroform:IAA (24:1) | Sigma | 25666-500ML | ||
Ethidium Bromide (EtBr) | Sigma | E1510-10ml | ||
Ethylenediaminetetraacetic acid disodium salt dihydrate | Sigma | E5134 | ||
Hybridization oven | Fisher Scientific | 13-247-10 | 37°C & 55°C | |
Lysis Buffer | 0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3) | |||
Lysozyme | Sigma | L6876-5G | 125 mg in 1000 μl TE | |
Microcon® Ultracel YM-30 Centrifugal Filter Unit | Millipore | 42410 | 50 bp cut off | |
Parafilm | Fisher | 13-374-10 | ||
Phenol:Chloroform:IAA (25:24:1) | Sigma | 77617-500ML | ||
Proteinase K | Qiagen | 19133 | ||
RNase A | Fisher | BP2539-100 | 10 μg/ml | |
Safe Imager™ Blue light transilluminator | Invitrogen | S37102 | ||
Sodium Dodecyl Sulfate (SDS) | Sigma | L4509 | 20% SDS | |
Sterivex filters | Fisher | SVG010RS | ||
Sucrose | Sigma | S0389 | ||
Table top centrifuge | Eppendorf | 5415D | ||
TE buffer, pH 8.0 | ||||
Trizma® base | Sigma | T1503 | ||
Ultracentrifuge rotor, TLA 100 | Beckman Coulter | 343847 | ||
Ultracentrifuge tube (7X20mm, PC) | Beckman | 343775 | ||
Ultracentrifuge tube rack | Beckman | 348302 | ||
Ultracentrifuge, Optima® Max | Beckman Coulter |