TITLE: COAST Biostic gDNA extraction using vacuum manifold
AUTHORS: holly.steininger

[DESCRIPTION]
COAST Extraction Protcol - HMS

[STEPS]
SECTION: DNA isolation
5. Secure the PowerBead Tube horizontally using the Vortex Adapter tube holder for the vortex (cat. no. 13000-V1-24). Vortex at maximum speed for 10 min.
due to limited vortex attachments you have to vortex in batches of 48 
SECTION: DNA isolation
6. Centrifuge the PowerBead Tube to pellet debris at 10.000 x g, 1 min. 
SECTION: DNA isolation
7. Add supernatant to the 2 ml deep well 96 well plate with pre aliquoted  100 µL of Solution IRS, seal, and vortex to mix. Incubate for 5 min at room temperature. Note: Longer incubation in Solution IRS does not affect DNA yield or purity (sample may be incubated up to 10 min in Solution IRS).

SECTION: DNA isolation
8. Centrifuge at 3.750 rpm, 3 min. 
 


SECTION: DNA isolation
9. Transfer the supernatant with a multichannel pipette to 2ml deep 96 well plate containing BB solution.  Pipette or pulse vortex to mix. Briefly centrifuge to collect any liquid from the top of the lid.

SECTION: DNA isolation
10. Place 96 well DNA column plate on a vacuum manifold and load 800 µLof lysate to the wells. Turn on vacuum and allow all liquid to pass through. Repeat this step until all the lysate has been loaded onto the DNA column (usually only x2).

SECTION: DNA isolation
11. Wash by adding 500 µL of Solution CB. Turn on vacuum and allow all liquid to pass through.

SECTION: DNA isolation
12. Wash with another 500 µL of Solution CB and Turn on vacuum and allow all liquid to pass through

SECTION: DNA isolation
13. Continue to allow vacuum to dry the DNA Column membrane. Approximately 2 min

SECTION: DNA isolation
14. Remove well plate and place over a 96-well semi skirted plate.
SECTION: DNA isolation
15. Elute by adding 50 µL of Solution EB directly in the center of the membrane. Allow sit at room temperature for up to 5 min to maximize the elution.

SECTION: DNA isolation
16. Centrifuge the stacked plates in the centrifuge at 3.750 x g, 2 min to elute DNA.
This step the membrane+ plate looks very suspect I covered the membrane in foil and used foil to 

secure the membrane to the semi skirt plate. 

 

SECTION: DNA isolation
17. Discard DNA column plate and aliquot 2 µL of eluted DNA into a second semi skirted 96 well plate.

SECTION: DNA isolation
3. Heat MBL solution at 55 °C for  5 min to  10 min.  Using a multichannel and a reservoir transfer 450 µL MBL solution to 2 mL PowerBead Tube. Add as much sample as possible per tube at a maximum amount of 1.3 mL (650x2).
Create 3 NTCs per extraction batch 
SECTION: DNA isolation
4. Vortex for10 sto mix and place in a 70 °C heat block or water bath for 15 min 
due to limited heat blocks you have to heat in batches of 48 
SECTION: Sample prep
2. Aliquot 100 µL of IRS solution into  each well a 2ml deep 96 well plate and seal with foil
Aliquot 1 mL BB solution into each well of a 2ml deep 96 well plate and seal with foil
Aliquot 700 µL EB solution in 8 2mL tubes 
Label 96 Powerbead tubes 1-93  (or sample number) and 3 control tubes on lid and side of the tube



SECTION: Sample prep
1. Place tubes on bin of ice for  approximately 60 min to thaw sample fully. Record all information present as you move the tubes.

SECTION: DNA isolation
18. Seal both plates containing 48 µL of eluted DNA and the other containing 2 µL with foil and store at -20 °C
Note: We recommend storing DNA frozen (–20° to –80°C) as Solution EB does not contain EDTA.