TITLE: QIAamp DNA FFPE Tissue Kit protocol
AUTHORS: SANTIAGO SEPULVEDA

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The QIAamp DNA FFPE Tissue Kit is optimized for purification of DNA from FFPE tissue</div><div class = "text-block">sections. It uses well-established QIAamp DNA Micro technology for purification of genomic</div><div class = "text-block">and mitochondrial DNA from small sample volumes or sizes. The kit combines the selective</div><div class = "text-block">binding properties of a silica-based membrane with flexible elution volumes of between</div><div class = "text-block">20 and 100 µl.</div><div class = "text-block">Specially optimized lysis conditions allow genomic DNA to be efficiently purified from FFPE</div><div class = "text-block">tissue sections without the need for overnight incubation. Incubation at an elevated temperature</div><div class = "text-block">after proteinase K digestion partially removes formalin crosslinking of the released DNA,</div><div class = "text-block">improving yields, as well as DNA performance in downstream assays. Note that DNA isolated</div><div class = "text-block">from FFPE samples is usually of lower molecular weight than DNA from fresh or frozen samples.</div><div class = "text-block">The degree of fragmentation depends on the type and age of the sample and the conditions</div><div class = "text-block">used for fixation.</div><div class = "text-block">After sample lysis, the simple QIAamp DNA Micro procedure, which is highly suited for</div><div class = "text-block">simultaneous processing of multiple samples, yields pure DNA in less than 30 minutes.</div><div class = "text-block">DNA is eluted in Buffer ATE or water and is immediately ready for use in amplification</div><div class = "text-block">reactions or for storage at –30°C to –15°C. Purified DNA is free of proteins, nucleases and</div><div class = "text-block">other impurities.</div></div>

[STEPS]
?. [Long incubations]
Incubate at  for  (or until the sample has been completely lysed)
56 °C
?. [Long incubations]
Incubate at  for
90 °C
Incubation at 90°C in Buffer ATL partially reverses formaldehyde modification of nucleic acids. Longer incubation times or higher incubation temperatures may result in more fragmented DNA. If using only one heating block, leave sample at room temperature after the 56°C incubation until the heating block has reached 90°C.
?. [Isolation and elution]
Briefly centrifuge the 1.5 ml tube to remove drops from the lid.
?. [Isolation and elution]
Allow the sample to cool to  before adding RNAse A. Add  and incubate for  at  before continuing with step 15.
0 Room temperature
2 µl
0 Room temperature
?. [Isolation and elution]
Add  of buffer  and mix thoroughly by vortexing. Then add  ethanol (96-100%) and mix again thoroughly by vortexing.
200 µl
200 µl
Buffer AL and ethanol can be premixed when working with multiple samples, e.g., 1 ml ethanol and 1 ml buffer AL for 10 samples. Is essential that the sample, buffer and ethanol are mixed immediately to yield an homogeneous solution. If done this way, you can vortex the samples for   -
?. [Isolation and elution]
Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the lid.
?. [Isolation and elution]
Carefully transfer the entire lysate to the QIAamp MinElute column (includes a 2 ml collection tube) without wetting the rim, close the lid and centrifuge at . Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through.
Centrifuge: 6000 34, 1 min
If the lysate has not completely passed through the membrane after centrifugation, centrifuge again at a higher speed until the QIAamp MinElute column is empty.
?. [Isolation and elution]
Carefully open the QIAamp MinElute column and add without wetting the rim. Close the lid and centrifuge at . Place the QIAamp MinElute column in a clean 2 ml collection tube and discard the collection tube containing the flow-through
500 µl
Centrifuge: 6000 34, 2 min
?. [Isolation and elution]
Carefully open the QIAamp MinElute column and add  without wetting the rim. Close the lid and centrifuge at   . Place the QIAamp MinElute column in a clean 2 ml collection tube and discard the collection tube containing the flow-through.
500 µl
Centrifuge: 6000 34, 150 s
Contact between the QIAamp MinElute column and the flow-through should be avoided. Seome centrifuge rotors may vibrate upon deceleration, resulting in the flow-through, which contains ethanol, coming into contact with the QIAamp MinElute column. Take care whem removing the QIAamp MinElute column and collection tube from the rotor, so that flow-through does not  come into contact with the QIAamp MinElute column.
?. [First section preparation]
Prepare the workspace and leavy at hand the (see the Before start section),  and ethanol  per sample. Other reagents will be used after the first 1 hour incubation.
1 mL
?. [FFPE slices processing]
Resuspend pellet in  of , add and mix by vortexing.
180 µl
20 µl
?. [FFPE slices processing]
Open the tube and incubate at   or   for   or until the residual ethanol has evaporated.
0 Room temperature
37 °C
?. [FFPE slices processing]
With the lid closed, invert the tube and flick the bottom to spread the ethanol volume across the tube.
?. [FFPE slices processing]
Remove supernatant by pipetting without removing any pellet, using  a fine pipet tip.
?. [FFPE slices processing]
Centrifuge
Centrifuge: 20000 34, 150 s
?. [FFPE slices processing]
Add  ethanol (96-100%) to the pellet and mix by vortexing.
1 mL
?. [FFPE slices processing]
Remove the supernatant by pipetting. Do not remove any of the pellet.
?. [FFPE slices processing]
and
Centrifuge: 20000 34, 150 s
?. [FFPE slices processing]
Add   Xylene to the sample, close the lid and shake vigorously for
1 mL
?. [Isolation and elution]
Centrifuge at
Centrifuge: 20000 34, 210 s
This step is completely necessary, as ethanol carryover may interfere with some downstream applications.
?. [Isolation and elution]
Place the QIAamp MinElute column in a clean 1.5 ml microcentrifuge tube (not provided), and discard the collection tube containing the flow-through. Carefully open the lid of the QIAamp MinElute column and apply  of Buffer ATE to the center of the membrane.
35 µl
IMPORTANT: Ensure the Buffer ATE is equilibrated at  . If using small elution volumes ( ), dispense the Buffer ATE onto the center of the membrane to ensure complete elution of bound DNA. Final volume of the eluate will be   less than the volume of elution applied to the column.
?. [Isolation and elution]
Close the lid and incubate at  for  . Centrifuge .
0 Room temperature
Centrifuge: 20000 34, 90 s
Incubation time can be as little as 1 minute, but 5 minutes has been observed to increase yield.
?. [Isolation and elution]
before incubation time ends, prepare and leave at hand 1.5 ml microcentrifuge tubes (1 per sample), 0.5 ml tubes (2 per sample), collection tubes (3 per sample), , ethanol (96-100%) (),, and Buffer ATE.
[per sample]
?. [Isolation and elution]
and aliquot samples for DNA quality tests and usage in 0.5 ml centrifuge tubes (If used in the next 24 hours, store at  , otherwise freeze at  - .
4 °C
-30 °C
-15 °C