Quick Guide for FormaPure Total

Be sure to read the full instructions for use before proceeding with this checklist. Video links are provided throughout the protocol demonstrating best practices. FormaPure.com

START HERE

1. Sample Preparation

☐ a. Place one to three 10 μm FFPE tissue sections into a 1.5 mL tube.


2. Deparaffinization

☐ a. Add 450 μL of mineral oil - [image: MO] - and immerse the tissue completely. FormaPure.com/oil
☐ b. Incubate at 80°C for 5 mintues.
☐ c. Vortex two times for 5 seconds.


3. Tissue Digestion

☐ a. Add 200 μL of Lysis buffer - [image: LBA]
☐ b. Centrifuge at 10,000 x g for 15 seconds.
☐ c. Add 20 μL of Proteinase K to the lower phase.
Pipette mix 10 times.
☐ d. Incubate at 60°C for 120 minutes.    Start __________    End __________   

 

The protocol splits at this point depending on which extractions you’d like to perform.

For RNA isolation only: Proceed to “RNA Only isolation”

For DNA isolation only: Proceed to “DNA Only isolation”

For Total isolation: Continue below

 

4. Lysate Splitting

☐ a. Centrifuge at 10,000 x g for 5 minutes.
☐ b. Transfer 100 μL of the Lysate (bottom layer) to a new tube or plate for RNA isolation. Avoid disturbing the upper phase (mineral oil) or any pellet that may be present.


The transferred 100 μL should proceed to:“Total Isolation - RNA”

The remaining content should proceed to: “Total Isolation - DNA”

Note: The RNA portion should be processed right away. The DNA portion can remain at 60°C up to overnight.


Total isolation - RNA

5. First Bind

☐ a. Add 150 μL of Bind buffer - [image: BBA] FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant

 

6. Ethanol Wash

☐ a. Remove samples from the magnet
☐ b. Add 375 μL of 80% ethanol.
☐ c. Pipette mix 20 times to resuspend the beads. FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes. FormaPure.com/dry


7. DNase I Treatment

☐ a. Add 80 μL of water.
☐ b. Add 10 μL of 10x DNase I buffer and 10 μL of DNase I and pipette mix 5 times.
☐ c. Incubate at 37°C for 20 minutes.


8. Re-bind

☐ a. Add 150 μL of Re-bind buffer - [image: RBA]
☐ b. Pipette mix 10 times
☐ c. Incubate for 5 minutes
☐ d. Separate on the magnet for 10 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant

 

9. Ethanol Wash

☐ a. Remove samples from the magnet
☐ b. Add 375 μL of 80% ethanol
☐ c. Pipette mix 20 times to resuspend the beads FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes FormaPure.com/dry

 

10. Elution

☐ a. Remove samples from the magnet
☐ b. Add 40 μL water
☐ c. Pipette mix 10 times to resuspend the beads FormaPure.com/resuspend
☐ d. Incubate at 60°C for 1 minute
☐ e. Separate on the magnet for 1 minute FormaPure.com/separate
☐ f. Transfer eluate to a new plate or tube
☐ g. Store at -20°C, or -80°C for long-term storage

 

Total isolation - DNA

5. Lysis

☐ a. If there is a pellet, pipette mix 10 times
☐ b. Incubate at 60°C for 60 minutes
(or up to overnight if needed)

 

6. Decrosslinking

☐ a. Incubate at 80°C for 60 minutes
☐ b. Transfer lysate to a new tube or plate

 

7. RNase Treatment

☐ a. Add 2.5 μL of RNase A.
☐ b. Pipette mix 5 times.
☐ c. Incubate for 5 minutes.

8. Bind DNA

☐ a. Add 150 μL of Bind buffer - [image: BBA] FormaPure.com/bind
☐ b. Pipette mix 10 times.
☐ c. Incubate for 5 minutes.
☐ d. Separate on the magnet for 10 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant

9. Wash

☐ a. Remove samples from the magnet.
☐ b. Add 200 μL of Wash buffer -[image: WBA]
☐ c. Pipette mix 15 times to resuspend the beads. FormaPure.com/resuspend
☐ d. Separate on the magnet for 10 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant


10. Ethanol Wash & Air Dry

☐ a. Remove samples from the magnet.
☐ b. Add 375 μL of 80% ethanol.
☐ c. Pipette mix 20 times to resuspend the beads. FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes. FormaPure.com/dry


11. Elution

☐ a. Remove samples from the magnet.
☐ b. Add 40 μL water.
☐ c. Pipette mix 10 times to resuspend the beads. FormaPure.com/resuspend
☐ d. Incubate at 60°C for 1 minute.
☐ e. Separate on the magnet for 1 minute. FormaPure.com/separate
☐ f. Transfer eluate to a new plate or tube.
☐ g. Store at -20°C.


RNA-only isolation

4. Lysis Transfer

☐ a. Centrifuge at 10,000 x g for 5 minutes. FormaPure.com/lysate
☐ b. Transfer all of the lysate to a new tube or plate. FormaPure.com/bind


5. First Bind

☐ a. Add 300 μL of Bind buffer - [image: BBA]
☐ b. Pipette mix 10 times.
☐ c. Incubate for 5 minutes.
☐ d. Separate on the magnet for 10 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant


6. Ethanol Wash

☐ a. Remove samples from the magnet.
☐ b. Add 750 μL of 80% ethanol.
☐ c. Pipette mix 20 times to resuspend the beads. FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes. FormaPure.com/separate
☐ e. Aspirate and discard supernatant. FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes. FormaPure.com/dry


7. DNase I Treatment

☐ a. Add 80 μL of water.
☐ b. Add 10 μL of 10x DNase I buffer and 10 μL of DNase I and pipette mix 5 times.
☐ c. Incubate at 37°C for 20 minutes.


8. Re-bind

☐ a. Add 150 μL of Re-bind buffer - [image: RBA]
☐ b. Pipette mix 10 times
☐ c. Incubate for 5 minutes
☐ d. Separate on the magnet for 10 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant


9. Ethanol Wash

☐ a. Remove samples from the magnet
☐ b. Add 750 μL of 80% ethanol
☐ c. Pipette mix 20 times to resuspend the beads FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes FormaPure.com/dry


10. Elution

☐ a. Remove samples from the magnet
☐ b. Add 40 μL water
☐ c. Pipette mix 10 times to resuspend the beads FormaPure.com/resuspend
☐ d. Incubate at 60°C for 1 minute
☐ e. Separate on the magnet for 1 minute FormaPure.com/separate
☐ f. Transfer eluate to a new plate or tube
☐ g. Store at -20°C, or -80°C for long-term storage


DNA-only isolation

4. Lysis

☐ a. If needed, extended 60°C lysis Start __________    End __________   

(up to overnight)


5. Decrosslinking

☐ a. Incubate at 80°C for 60 minutes Start __________    End __________   

☐ b. Transfer all of the lysate to a new tube or plate FormaPure.com/lysate


6. RNase Treatment

☐ a. Add 5 μL of RNase A
☐ b. Pipette mix 5 times
☐ c. Incubate for 5 minutes


7. Bind DNA

☐ a. Add 300 μL of Bind buffer - [image: BBA] FormaPure.com/bind
☐ b. Pipette mix 10 times
☐ c. Incubate for 5 minutes
☐ d. Separate on the magnet for 10 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant


8. Wash

☐ a. Remove samples from the magnet
☐ b. Add 400 μL of Wash buffer - [image: WBA]
☐ c. Pipette mix 15 times to resuspend the beads FormaPure.com/resuspend
☐ d. Separate on the magnet for 10 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant


5. Ethanol Wash & Air Dry

☐ a. Remove samples from the magnet
☐ b. Add 750 μL of 80% ethanol
☐ c. Pipette mix 20 times to resuspend the beads FormaPure.com/resuspend
☐ d. Separate on the magnet for 3 minutes FormaPure.com/separate
☐ e. Aspirate and discard supernatant FormaPure.com/supernatant
☐ f. Air dry on the magnet for 10 minutes FormaPure.com/dry


10. Elution

☐ a. Remove samples from the magnet
☐ b. Add 40 μL water
☐ c. Pipette mix 10 times to resuspend the beads FormaPure.com/resuspend
☐ d. Incubate at 60°C for 1 minute
☐ e. Separate on the magnet for 1 minute FormaPure.com/separate
☐ f. Transfer eluate to a new plate or tube
☐ g. Store at -20°C