The ZR FFPE DNA MiniPrep™ 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.
| Step 1 |
To a deparaffinized tissue sample (≤25 mg) in a microcentrifuge tube, add the following:
| H2O |
45µl |
| 2X Digestion Buffer |
45µl |
| Proteinase K |
10µl |
Note: If your 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 3 & 4.
|
| Step 2 |
| Rapid Digenstion |
Standard Digestion |
| Incubate at 55°C for 1-4 hours |
Incubate at 55°C overnight (12-16 hrs) |
Note: The Rapid Digestion is recommended for processing slide tissue sections but should also besufficient for most tissue samples. However, the Standard Digestion ensures maximum yields of DNA from even tough-to-lyse (collagen-rich, fibrous, etc.) tissue samples.
|
| Step 3 |
Transfer the digestion to 94°C and incubate for 20 minutes. When done add 5 µl of RNase A, mix, and then incubate an additional 5 minutes at room temperature.
Note: It is recommended to skip the heat treatment in Step 3 and the addition of isopropanol in Step 4 if only double stranded DNA is required (e.g. OneStep qMethyl™ Array, Cat. No. D5312).
|
| Step 4 |
Add 350 µl of Genomic Lysis Bufferto the tube and mix thoroughly by vortexing.
| To Isolate Total DNA >50 bp |
To Isolate DNA >500 bp |
|
Add 135 µl of isopropanol* (user
supplied) to the sample, mix
thoroughly, and proceed to Step 5
|
Proceed directly to Step 5 |
Note: When working with a new sample, it is recommended to isolate total DNA as a precaution. FFPE DNA may be highly degraded and DNA >500 bp may not be present in sample.
|
| Step 5 |
Centrifuge at 10,000 x g for 1 minute to remove insoluble debris and then transfer the supernatant to aZymo-Spin™ IIC Column in a Collection Tube. Centrifuge at 10,000 x g for 1 minute.
|
| Step 6 |
Add 200 µl of DNA Pre-Wash Buffer to the spin column in a new Collection Tube. Centrifuge at 10,000 x g for 1 minute.
|
| Step 7 |
Add 400 µl of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for one 1 minute.
|
| Step 8 |
Transfer the Zymo-Spin™ IIC Column to a clean microcentrifuge tube. Add ≥50 µl DNA Elution Buffer or water (e.g., add ≥100 µl if sampling 25 mg tissue) to the spin column. Incubate 2-5 minutes at room temperature, then centrifuge at top speed for 30 seconds to elute the DNA. The eluted DNA can be used immediately for molecular based applications or stored ≤-20ºC for future use.
|
For specific notes and additional information, please see the product protocol PDF.
