DNA Extraction Protocol from Sterivex Filters

Clean bench area with bleach, ethanol, and RNase away

Turn on incubator and set to 56°C

Wipe down 1000 µL and 200 µL pipettes with RNase Away and UV for 15 minutes on each side

Assemble materials and reagents needed:

• 1000 µL pipette tips (that fit in the inlet of the Sterivex)
• 200 µL pipette tips
• Proteinase K (from Qiagen DNeasy Blood & Tissue Kit)
• ATL Buffer (from Qiagen DNeasy Blood & Tissue Kit)

Remove sterivex filters from -15°C freezer

For each filter:

Remove Sterivex from Whirl-Pak bag and remove cap from inlet end of Sterivex filter

Slowly pipette 80 µL of Proteinase K directly on top of the filter through the inlet, avoiding backsplash by expelling slowly

Slowly pipette 720 µL of ATL buffer directly on top of the filter through the inlet, again avoiding backsplash

Secure Sterivex with same luer cap

Handshake vigorously for several seconds

Place all filters onto roller shaker in incubator

Incubate at 56°C for ~24 hours (minimum of 12 hours; try to incubate for the same amount of time for all filters for a particular project) while rotating at approximately 6 rpm

Clean bench area (including vortex), centrifuge area, and centrifuge with bleach, ethanol, and RNase away.

Soak all tube racks in a 10% bleach solution, followed by 3 rinses with DI water. Let dry and UV for 15 minutes

UV Qubit and LoBind tubes for 15 minutes (in pre-sterilized tube racks) and label with sample numbers:

• Qubit: (# of samples+ extraction blank) + 2 for standards
• 1.5/2 mL LoBind: (# of samples + extraction blank) * 3
• 5 mL LoBind: # of samples + extraction blank
• Enough tubes (any type, does not need to be LoBind) to hold AE Buffer that fit in heat block (see Step 15), and to hold Qubit working solution (see Step 23.1)

Open and label additional tubes needed from Qiagen DNeasy Blood & Tissue Kit:

• Packaged spin columns: # of samples + extraction blank
• Additional collection tubes: (# of samples + extraction blank) * 2

Wipe down 1000 µl pipette, 200 µl pipette, 100 µl pipette, 10 µl pipette with RNase Away and UV for 15 minutes on each side

Place 50 mL tube of molecular-grade ethanol in freezer or on ice to chill for later use

Heat aliquot (volume calculation below) of AE buffer (from Qiagen DNeasy Blood & Tissue Kit) on heating block at 70°C

Volume: (# of samples including blank * 100 µl) * 1.1

Make cryo-labels for storage tubes (2 of 3 1.5/2 mL LoBind tubes already sterilized)

Assemble other reagents & materials needed:

• Sterile 3 mL syringes (one per sample)
• AL Buffer (from Qiagen DNeasy Blood & Tissue Kit)
• Buffer AW1 (from Qiagen DNeasy Blood & Tissue Kit)
• Buffer AW2 (from Qiagen DNeasy Blood & Tissue Kit)

Remove Sterivex filters from incubator

Remove liquid from each filter:

Handshake vigorously for several seconds

Remove caps from filter

Using a sterile 3 mL syringe attached to the inlet end of the Sterivex, remove all liquid from filter, record the volume, and transfer into a 5 mL LoBind tube

Create an extraction blank by adding 1000 uL nuclease free water subbed in for extracted liquid to a 5 mL LoBind tube

For each sample and extraction blank, add AL buffer and 0°C ethanol to the extracted liquid in a 1:1:1 ratio and vortex vigorously for 10 seconds

For each sample and extraction blank, filter the mixture through a spin column:

Pipet the mixture (650 µl at a time) into a DNeasy Mini Spin column placed in a collection tube

Spin in micro-centrifuge for 1 minute at 6000 x g (8000 rpm)

Discard flow throw, and dab the rim of the spin column dry on a Kimwipe

Repeat sub-steps of 22 until all sample is filtered through spin column

For each sample and extraction blank follow the remaining Qiagen DNeasy Tissue and Blood Kit steps:

Place the spin column in a new collection tube, add 500 µl Buffer AW1

Immediately transfer tubes to room temperature

Incubate at room temperature for 10 minutes

Centrifuge for 1 min at 6000 x g (8000 rpm)

Re-elute DNA from DNA LoBind tube (apply eluate back on spin column while tubes are in heat block)

Incubate at room temperature for 10 minutes

Centrifuge for 1 min at 6000 x g (8000 rpm)

Discard the spin column

Centrifuge for 1 minute at 6000 x g (8000 rpm). Discard flow through and collection tube.

Place in new collection tube and add 500 µl Buffer AW2

Centrifuge for 3 min at 20,000 x g (14000 rpm) to dry the membrane

Discard flow through and dab rim of spin column on a clean Kimwipe

Place the spin column back in collection tube and centrifuge for 1 min at 20,000 x g (14000 rpm)

Transfer spin column to new 1.5/2 mL LoBind tube with cap left open

Place samples in 70°C heat block

Add 100 µl Buffer AE (4 samples at a time) directly over membrane

Aliquot 1 µl of each sample into labeled Qubit tubes

Transfer ~50 µl of remaining DNA extract to 2 pre-marked DNA LoBind tubes with lids intact:

• 1 archive tube to store at -80°C
• 1 working tube to store at -15°C
  Note

  Aliquot to archive tube first, so that archive volumes are consistently 50 µl. Working tube volumes may be slightly less than 50 µl due to Qubit aliquot, etc.

Use Qubit to measure DNA concentrations in all samples:

Make Qubit working solution:

• Reagent: ((# of samples + extraction blank + 2) * 1 )*1.1
• Buffer: ((# of samples + extraction blank + 2) * 199)*1.1

Vortex Qubit working solution

Add 199 µl of working solution to Qubit tubes containing sample DNA

Add 190 µl of working solution to Qubit tubes for 2 standards

Add 10 µl of respective standards to Qubit tubes for 2 standards

Mix all tubes gently by vortexing, being sure not to introduce bubbles

Allow tubes to incubate at room temperature for 2 minutes

Read samples with Qubit fluorometer and record DNA concentrations