Dec 21, 2016
Quick-DNA™ FFPE Kit Protocol
- Zymo Research
- Zymo Research - The Epigenetics Company
External link: https://www.zymoresearch.com/dna/genomic-dna/solid-ffpe-tissue-dna/quick-dna-ffpe-kit/quick-dna-ffpe-kit
Protocol Citation: Zymo Research 2016. Quick-DNA™ FFPE Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.giybufw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 25, 2016
Last Modified: March 21, 2018
Protocol Integer ID: 4408
Abstract
The Quick-DNA™ FFPE Kit provides a simple and reliable method for high yield/quality DNA isolation from formalin-fixed, paraffin embedded (FFPE) tissue samples and sections. The unique chemistries of the product have been optimized for maximum recovery of non-crosslinked, ultra-pure DNA without RNA contamination. Simply digest deparaffinized tissues using the provided Proteinase K, heat, and then purify the DNA with the Fast-Spin columns in the kit. DNA >50 bp or >500 bp can be selectively isolated by altering the lysis buffer conditions as given in the protocol. PCR inhibitors are effectively removed during the isolation procedure, and eluted DNA is ideal for PCR, Next-Gen library prep, enzymatic manipulation, etc.
Guidelines
Product Contents
1 The Proteinase K is stable as shipped. Add 260 µl Proteinase K Storage Buffer to each Proteinase K tube prior to use. The final concentration of Proteinase K after the addition of Proteinase K storage Buffer is ~20 mg/ml. Store at -20° C.
2 Recommended: Add beta-mercaptoethanol to 0.5(v/v) i.e., 250 µl per 50 ml or 500 µl per 100 ml.
3 Before starting, add 48 ml 100% ethanol (52 ml 95% ethanol) to the 12 ml Genomic DNA Wash 2 concentrate.
4 Re-suspend lyophilized RNase A in 300 µl of ddH2O. Store at 4° C.
Specifications
Sample Size Up to 25 mg tissue from paraffin block or up to four (4) tissue sections (≤20 µm thick) with a total surface area ~20 cm2 . It is recommended to use 1-2 sections if performing the protocol for the first time. Compatible with fresh/frozen tissue specimens.
DNA Recovery High quality total DNA (A260/A280 >1.8) can be eluted into small volumes (i.e., ≥25 µl) allowing for highly concentrated samples. The maximum DNA binding capacity of the provided spin column is ~25 µg.
Processing Time As little as 4 hours when processing large amounts of tissue. For maximum yields of the highest quality DNA, it is recommended to process samples overnight.
Equipment/Reagents Microcentrifuge, thermomixer or heat block/bath capable of 55°C and 90°C, isopropanol, beta-mercaptoethanol (optional).
Product Description
The Quick-DNA™ FFPE Kit provides a simple and reliable method for high yield/quality DNA isolation from formalin-fixed, paraffin embedded (FFPE) tissue samples and sections. The unique chemistries of the product have been optimized for maximum recovery of non-crosslinked, ultra-pure DNA without RNA contamination. Simply digest deparaffinized tissues using the provided Proteinase K, heat, and then purify the DNA with the Fast-Spin columns in the kit. DNA >50 bp or >500 bp can be selectively isolated by altering the lysis buffer conditions as given in the protocol. PCR inhibitors are effectively removed during the isolation procedure, and eluted DNA is ideal for PCR, Next-Gen library prep, enzymatic manipulation, etc. Shown below is a schematic and performance overview of the procedure.
Buffer Preparation
Add 260 µl Proteinase K Storage Buffer to reconstitute lyophilized Proteinase K at 20 mg/ml. Vortex to dissolve. Store at -20° C.
Before starting, add 48 ml 100% ethanol (52 ml 95% ethanol) to the 12 ml Genomic DNA
Wash 2 concentrate.
Resuspend lyophilized RNase A in 300 µl of ddH20. Store at 4° C.
Recommended: Add beta-mercaptoethanol (user supplied) to the Genomic Lysis Buffer to a final dilution of 0.5%(v/v) i.e., 250 µl per 50 ml.
Materials
MATERIALS
Quick-DNA™ FFPE KitZymo ResearchCatalog #D3067
Before start
Buffer Preparation
Add 260 μl Proteinase K Storage Buffer to reconstitute lyophilized Proteinase K at 20 mg/ml. Vortex to dissolve. Store at -20° C.
Before starting, add 48 ml 100% ethanol (52 ml 95% ethanol) to the 12 ml Genomic DNA
Wash 2 concentrate.
Resuspend lyophilized RNase A in 300 μl of ddH20. Store at 4° C.
Recommended: Add beta-mercaptoethanol (user supplied) to the Genomic Lysis Buffer to a final dilution of 0.5%(v/v) i.e., 250 μl per 50 ml.
Deparaffinization
Deparaffinization
Remove (trim) excess paraffin wax from sample and transfer the sample to a 1.5 ml microcentrifuge tube.
Note
Note: Up to 25 mg tissue from a paraffin block or up to four (4) tissue sections (≤20 μm thick) with a 2 total surface area ~20 cm . It is recommended to use start with 1-2 sections.
Note
Note: If using fresh/frozen tissue specimens proceed directly with Proteinase K Digestion & DNA Isolation.
Add 400 μl of Deparaffinization Solution to the sample. Incubate at 55°C for 1 minute. Vortex briefly.
00:01:00
Note
Remove Deparaffinization Solution from the sample and proceed to next section.
Tissue Digestion
Tissue Digestion
To the deparaffinized tissue sample (≤ 25 mg) in a microcentrifuge tube, add the following mixture:
| H2O | 45μl | |
| 2X Digestion Buffer | 45μl | |
| Proteinase K | 10μl |
Note
Note: If the tissue sample is too large for the digestion volume, scale up the digestion to 200 μl while keeping the amount of Proteinase K the same. Double the reagent volumes indicated in Step 8 & 9 of the DNA Purification section.
Digest 1-4 hours or overnight at 55 °C.
| Rapid Digestion | Standard Digestion | |
| Incubate at 55°C for 1-4 hours | Incubate at 55°C overnight (12-16 hrs) |
Note
Note: The Rapid Digestion is recommended for processing slide tissue sections. The Standard Digestion ensures maximum yields of DNA from tough-to-lyse (collagen-rich, fibrous, etc.) or large tissue samples.
Transfer the digestion to 94°C and incubate for 20 minutes.
00:20:00
Once done, add 5 μl of RNase A, mix, and incubate an additional 5 minutes at room temperature.
00:05:00
DNA Purification
DNA Purification
Add 350 μl of Genomic Lysis Buffer to the tube and mix thoroughly by vortexing.
Add 135 μl of isopropanol1 (user supplied) to the sample and mix thoroughly.
Note
1 ssDNA will also be purified if present in the sample upon the addition of isopropanol.
This procedure will isolate total DNA > 50 bp. To isolate only DNA > 500 bp, skip this step.
FFPE DNA may be highly degraded and DNA >500 bp may not be present in sample.
Centrifuge at ≥ 12,000 x g for 1 minute to remove insoluble debris.
00:01:00
Note
This procedure will isolate total DNA > 50 bp. To isolate only DNA > 500 bp, skip this step.
Transfer the supernatant to a Zymo-Spin™ IIC Column2 in a Collection Tube.
Note
The maximum loading volume for the Zymo-Spin™ Column is ~700 μl.
Centrifuge at 10,000 x g for 1 minute.
00:01:00
Add 400 μl of Genomic DNA Wash 1 to the spin column in a new Collection Tube.
Centrifuge at 10,000 x g for 1 minute. Discard the flow-through.
00:01:00
Add 700 μl of Genomic DNA Wash 2 to the spin column.
Centrifuge at ≥ 12,000 x g for 1 minute. Discard the flow-through.
00:01:00
Add 200 μl of Genomic DNA Wash 2 to the spin column.
Centrifuge at ≥ 12,000 x g for 1 minute.
00:01:00
Transfer the Zymo-Spin™ IIC Column to a clean microcentrifuge tube. Add ≥ 50 μl DNA Elution Buffer3 or water (add ≥100 μl if sampling 25 mg tissue) to the spin column.
Note
3 Elution of DNA from the column is dependent on pH and temperature. If water is used, ensure the pH is >6.0. Also, the total yield may be improved by eluting the DNA with Elution Buffer or water pre-equilibrated to 60-70° C or by performing and pooling sequential elutions.
Incubate 2-5 minutes at room temperature.
00:05:00
Centrifuge at top speed for 30 seconds to elute the DNA.
00:00:30
The eluted DNA can be used immediately for molecular based applications or stored ≤-20°C for future use.