Preparation of Tara Sample DNA From Iron - Chloride Precipitates
Locate samples and record inventory number and number of filters ( or portions ) used .
Turn the filters precipitate - side out with bleached forceps that have been rinsed with water and use aluminum foil squares as work surface , discarding after each filter .
Remove filter from 50cc tube and refold so that precipitate side comes in contact with resuspension buffer Return filter to tube using forceps and sterile applicator sticks .
Keep filters dark and refrigerated until ready to suspend .
Prepare 1x or 2x resuspension buffer .
Prepare 20mL 1x or 10mL 2x per filter from 20L of seawater .
Add resuspension buffer to each filter Parafilm the tubes and wrap all of them in aluminum foil .
Put foil pack of tubes on rotator , in cold room , using rubber bands to secure , and rotate slowly overnight .
To recover resuspended viruses , remove the liquid at the bottom using a 5mL pipet and transfer to a fresh 15mL or 50mL sterile , labeled tube .
Using bleached and rinsed forceps or sterile applicator sticks , pull edge of filter up and over lip of tube and secure with the lid .
Centrifuge 1000rpm , 5min , 18°C to recover liquid left on filter .
Remove liquid and add more buffer if filter still has a lot of precipitate clinging to it .
Rotate for several more hours .
Repeat removal of liquid .
Dilute stock DNase 1 : 100 in 10x DNase buffer ( concentration = 400 U / mL ) .
Add 1 / 10th volume of diluted DNase to each sample Parafilm tubes , wrap them in aluminum foil and attach to tube rotator with rubber bands .
Incubate by rotating slowly at room temperature for 2 hours .
Inactivate DNase by adding EDTA and EGTA to 0.1M final concentration each .
Mix by inverting the tube several times .
Add the DNase treated and inactivated sample to the top reservoir of Amicon Ultra 100kDA centrifugal concentrators .
Centrifuge the concentrators at 1000 g for 5 minute intervals at 18°C until samples are at less than 2mL each .
Put 1mL of resin on one Wizard Prep column .
Thoroughly resuspend resin by shaking vigorously .
Mix 1mL per 0.5 - 1.0mL of DNA .
Add each 1mL of resin to a 3mL luer lock syringe attached to a Wizard column .
Push through into a 5mL snap - cap tube .
Save this tube until DNA quantification .
Remove syringe from column Remove plunger .
Reattach syringe barrel to column .
Wash columns with 2mL 80 % isopropanol pushing through with the syringe barrel into a waste container .
Put columns into 1.5mL centrifuge tube and centrifuge at 10,000 g for 2.5 minutes to remove residual alcohol .
Discard tube .
Place column into a fresh 1.5mL centrifuge tube and pipet on 50 - 100µL of TE ( 0.02µm filtered and heated to 80°C ) .
Vortex gently ( 1400rpm ) and let sit 1 minute .
Centrifuge at 10,000 g for 1 minute .
Transfer extracted DNA to Lo - bind DNA 0.5mL tubes .
Repeat elution above ( steps 33 - 36 ) one more time .
Use 1 - 2µL of the first elution and 4 - 5µL of the second elution for the Pico Green assay to quantify the extracted DNAQuant - iT dsDNA Pico Green assay kit ( Invitrogen ) .
Use the Excel spreadsheet to calculate the ng / µL DNA for each sample .
Store in - 80°C ultra - low freezer .
Label tubes with date , station , depth and concentration and store in - 80°C ultra - low freezer
