50 prep

Introduction and Overview

Introduction

The E.Z.N.A.® Universal Pathogen Kit allows rapid and reliable isolation of high-quality host genomic DNA, gram positive and negative bacterial DNA, fungal spore DNA, and viral DNA and RNA from tissue, urine, serum, and fecal samples. Elution volumes as low as 15 µL can be used to maintain higher nucleic acid concentrations.

The system combines the Omega Bio-tek’s MicroElute® LE DNA Columns with RBB Buffer to eliminate PCR inhibiting compounds within the samples and elute highly concentrated DNA. Purified DNA is suitable for PCR, restriction digestion, and hybridization applications. There are no organic extractions thus reducing plastic waste and hands-on time and multiple samples can be processed in parallel.

Kit Contents

Product Number	D4035-00	D4035-01
Purifications	5 preps	50 preps
Disruptor Tubes	5	50
MicroElute® LE DNA Columns	5	50
2 mL Collection Tubes	10	100
SLX-Mlus Buffer 5 mL 50 mL	5 mL	50 mL
DS Buffer	500 µL	4 mL
PCP Buffer	1.2 mL	12 mL
RBB Buffer	4 mL	40 mL
HBC Buffer	3 mL	21 mL
DNA Wash Buffer	1.5 mL	15 mL
Elution Buffer	2 mL	15 mL
Proteinase K Solution	150 µL	1.5 mL
User Manual

 

Storage and Stability

All of the E.Z.N.A.® Universal Pathogen Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows. Proteinase K Solution can be stored at room temperature for up to 6 months. For long-term storage, store Proteinase K Solution at 2-8°C. All remaining components should be stored at room temperature. During shipment or storage in cool ambient conditions, precipitates may form in some of the buffers. Dissolve such deposits by warming the solution at 37°C and gently shaking.

Preparing Reagents

1. Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.

Kit	100% Ethanol to be Added
D4035-00	6 mL
D4035-01	60 mL

2. Dilute HBC Buffer with 100% isopropanol follows and store at room temperature.

Kit	100% Isopropanol to be Added
D4035-00	1.2 mL
D4035-01	8.4 m

E.Z.N.A.® Universal Pathogen Kit - Tissue Protocol

E.Z.N.A.® Universal Pathogen Kit - Tissue Protocol

Materials and Equipment to be Supplied by User:

• Centrifuge capable of at least 12,000 x g

• Incubator capable of 70°C

• Centrifuge tubes with a capacity of at least 1.7 mL

• Nuclease-free 1.5 mL centrifuge tubes for DNA storage

• Vortexer

• 100% ethanol

• 100% isopropanol

• Optional: Mixer mill such as a SPEX CertiPrep Geno/Grinder® 2010 or Qiagen TissueLyser

Before Starting:

• Prepare HBC Buffer and DNA Wash Buffer according to the “Preparing Reagents” section on Page 4.

• Set an incubator to 70°C.

• Heat Elution Buffer to 70°C.

1. Briefly spin the Disruptor Tube to remove any glass beads from the wall of the tube. Uncap the Disruptor Tube and save the cap for use in Step 3.

2. Add 25-30 mg tissue.

3. Add 725 µL SLX-Mlus Buffer. Seal the Disruptor Tube with the cap removed in Step 1.

4. Vortex at maximum speed for 3-5 minutes to lyse and homogenize the samples. For best results, a Mixer Mill, such as Spex CertiPrep Geno/Grinder® 2010 or Qiagen Tissuelyser, should be used.

Note: Depending on the sample type and amount, the volume of SLX-Mlus Buffer may need to be adjusted so that 300 µL can be recovered during Step 12.

5. Centrifuge at 1,000-2,000 x g for 15 seconds at room temperature.

6. Uncap the Disruptor Tube and save the cap for use in Step 8.

7. Add 72 µL DS Buffer and 20 µL Proteinase K Solution.

8. Seal the Disruptor Tube with the cap removed in Step 6.

9. Vortex for 60 seconds to mix thoroughly.

10. Incubate at 70°C for 15 minutes. Mix once during incubation.

11. Centrifuge at 12,000 x g for 5 minutes.

12. Transfer 300 µL cleared supernatant to a 1.5 mL centrifuge tube (not provided).

Note: Do not transfer any debris as it can reduce yield and purity.

13. Add 600 µL RBB Buffer. Vortex to mix thoroughly.

14. Let sit at room temperature for 5 minutes.

15. Insert a MicroElute® LE DNA Column into a 2 mL Collection Tube.

16. Transfer 700 µL sample from Step 14 to the MicroElute® LE DNA Column.

17. Centrifuge at maximum speed for 1 minute.

18. Discard the filtrate and reuse the collection tube.

19. Transfer the remaining lysate from Step 14 to the MicroElute® LE DNA Column.

20. Centrifuge at maximum speed for 1 minute.

21. Discard the filtrate and reuse the collection tube.

22. Add 500 μL HBC Buffer. Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

23. Centrifuge at maximum speed for 30 seconds.

24. Discard the filtrate and collection tube.

25. Insert the MicroElute® LE DNA Column into a new 2 mL Collection Tube.

26. Add 700 μL DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions.

27. Centrifuge at maximum speed for 30 seconds.

28. Discard the filtrate and reuse the collection tube.

29. Repeat Steps 26-28 for a second DNA Wash Buffer wash step.

30. Centrifuge the empty MicroElute® LE DNA Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

31. Transfer the MicroElute® LE DNA Column into a nuclease-free 1.5 mL microcentrifuge tube (not provided).

32. Add 15-100 μL Elution Buffer heated to 70°C.

33. Let sit at room temperature for 2 minutes.

34. Centrifuge at maximum speed for 1 minute.

35. Repeat Steps 32-34 for a second elution step.

Note: Each 200 μL elution will typically yield of 60-70% of the DNA bound to the column. Two elutions will generally yield ~90%. However, increasing the elution volume will reduce the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 50-100 μL Elution Buffer which slightly reduces overall DNA yield. Volumes lower than 50 μL greatly reduce yields. In some instances yields may be increased by incubating the column at 70°C (rather than room temperature) upon the addition of Elution Buffer.

36. Store eluted DNA at -20°C.

E.Z.N.A.® Universal Pathogen Kit - Serum & Fecal Protocol

E.Z.N.A. Universal Pathogen Kit - Serum & Fecal Protocol

The following will isolate DNA and RNA from gram-positive & negative bacteria, fungal spores, and viruses. If only gram-negative bacteria or viral RNA & DNA is required, Steps 4-6 can be skipped.

Materials and Equipment to be Supplied by User:

• Centrifuge capable of at least 12,000 x g

• Incubator capable of 70°C

• Centrifuge Tubes with a capacity of at least 1.7 mL

• 1.5 mL centrifuge tubes for DNA storage

• Vortexer

• 100% ethanol

• 100% isopropanol

• Optional: Mixer mill such as a SPEX CertiPrep Geno/Grinder® 2010 or Qiagen TissueLyser

Before Starting:

• Prepare HBC Buffer and DNA Wash Buffer according to the “Preparing Reagents” section on Page 4.

• Set an incubator to 70°C.

• Heat Elution Buffer to 70°C.

1. Briefly spin the Disruptor Tube to remove any glass beads from the wall of the tube. Uncap the Disruptor Tube and save the cap for use in Step 3.

2. Add 250 µL fecal sample or serum.

3. Add 475 µL SLX-Mlus Buffer. Seal the Disruptor Tube with the cap removed in Step 1.

Note: If only gram-negative bacteria or viral RNA & DNA is required, Steps 4-6 can be skipped. Continue to Step 7 below.

4. Vortex at maximum speed for 3-5 minutes to lyse and homogenize samples. For best results, a Mixer Mill, such as Spex CertiPrep Geno/Grinder® 2010 or Qiagen Tissuelyser, should be used.

Note: Depending on the sample type and amount, the volume of SLX-Mlus Buffer may need to be adjusted so that 300 µL can be recovered during Step 12.

5. Centrifuge at 1,000-2,000 x g for 15 seconds at room temperature.

6. Uncap the Disruptor Tube and save the cap for use in Step 8.

7. Add 72µL DS Buffer and 20 µL Proteinase K Solution.

8. Seal the Disruptor Tube with the cap removed in Step 6.

9. Vortex for 60 seconds to mix thoroughly.

10. Incubate at 70°C for 15 minutes. Mix once during incubation.

11. Centrifuge at 12,000 x g for 5 minutes.

12. Transfer 300 µL cleared supernatant to a 1.5 mL centrifuge tube (not provided)

Note: Do not transfer any debris as it can reduce yield and purity.

13. Add 600 µL RBB Buffer. Vortex to mix thoroughly.

14. Let sit at room temperature for 5 minutes.

15. Insert a MicroElute® LE DNA Column into a 2 mL Collection Tube.

16. Transfer 700 µL sample from Step 14 to the MicroElute® LE DNA Column.

17. Centrifuge at maximum speed for 1 minute.

18. Discard the filtrate and reuse the collection tube.

19. Transfer the remaining lysate from Step 14 to the MicroElute® LE DNA Column.

20. Centrifuge at maximum speed for 1 minute.

21. Discard the filtrate and reuse the collection tube.

22. Add 500 μL HBC Buffer.

Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

23. Centrifuge at maximum speed for 30 seconds.

24. Discard the filtrate and collection tube.

25. Insert the MicroElute® LE DNA Column into a new 2 mL Collection Tube.

26. Add 700 μL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions. 27. Centrifuge at maximum speed for 30 seconds.

28. Discard the filtrate and reuse the collection tube.

29. Repeat Steps 26-28 for a second DNA Wash Buffer wash step.

30. Centrifuge the empty MicroElute® LE DNA Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

31. Transfer the MicroElute® LE DNA Column into a nuclease-free 1.5 mL microcentrifuge tube (not provided).

32. Add 15-100 μL Elution Buffer heated to 70°C.

33. Let sit at room temperature for 2 minutes.

34. Centrifuge at maximum speed for 1 minute.

35. Repeat Steps 32-34 for a second elution step.

Note: Each 200 μL elution will typically yield of 60-70% of the DNA bound to the column. Two elutions will generally yield ~90%. However, increasing the elution volume will reduce the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 50-100 μL Elution Buffer which slightly reduces overall DNA yield. Volumes lower than 50 μL greatly reduce yields. In some instances yields may be increased by incubating the column at 70°C (rather than room temperature) upon the addition of Elution Buffer.

36. Store eluted DNA at -20°C.

E.Z.N.A.® Universal Pathogen Kit - Urine & Blood Protocol

Mag-Bind® Universal Pathogen 96 Kit - Urine & Blood Protocol

The following will isolate DNA and RNA from gram-positive & negative bacteria, fungal spores, and viruses. If only gram-negative bacteria and viral RNA & DNA is required Steps 4-6 can be skipped.

Materials and Equipment to be Supplied by User:

• Centrifuge capable of at least 12,000 x g

• Incubator capable of 70°C

• Centrifuge Tubes with a capacity of at least 1.7 mL

• 1.5 mL centrifuge tubes for DNA storage

• Vortexer

• Ice bucket

• 100% ethanol

• 100% isopropanol

• Optional: Mixer mill such as a SPEX CertiPrep Geno/Grinder® 2010 or Qiagen TissueLyser

Before Starting:

• Prepare HBC Buffer and DNA Wash Buffer according to the “Preparing Reagents” section on Page 4

• Set an incubator to 70°C

• Heat Elution Buffer to 70°C

• Prepare an ice bucket

1. Briefly spin the Disruptor Tube to remove any glass beads from the wall of the tube. Uncap the Disruptor Tube and save the cap for use in Step 3.

2. Add 250 µL urine or whole blood sample to the Disruptor Tube.

3. Add 275 µL SLX-Mlus Buffer. Seal the Disruptor Tube with the cap removed in Step 1.

Note: If only gram-negative bacteria or viral RNA & DNA is required, Steps 4-6 can be skipped. Continue to Step 7 below.

4. Vortex at maximum speed for 3-5 minutes to lyse and homogenize samples. For best results, a Mixer Mill, such as Spex CertiPrep Geno/Grinder® 2010 or Qiagen Tissuelyser, should be used.

Note: Depending on the sample type and amount, the volume of SLX-Mlus Buffer may need to be adjusted so that 300 µL can be recovered during Step 13.

5. Centrifuge at 1,000-2,000 x g for 15 seconds at room temperature.

6. Uncap the Disruptor Tube and save the cap for use in Step 8.

7. Add 50 µL DS Buffer and 20 µL Proteinase K Solution.

8. Seal the Disruptor Tube with the cap removed in Step 6.

9. Vortex for 60 seconds to mix thoroughly.

10. Incubate at 70°C for 15 minutes. Mix once during incubation.

11. Add 200 µL PCP Buffer. Let sit on ice for 5 minutes.

12. Centrifuge at 12,000 x g for 10 minutes.

13. Transfer 300 µL cleared supernatant to a 1.5 mL centrifuge tube (not provided).

Note: Do not transfer any debris as it can reduce yield and purity.

14. Add 600 µL RBB Buffer. Vortex to mix thoroughly.

15. Let sit at room temperature for 5 minutes.

16. Insert a MicroElute® LE DNA Column into a 2 mL Collection Tube.

17. Transfer the 700 µL sample from Step 15 to the MicroElute® LE DNA Column.

18. Centrifuge at maximum speed for 1 minute.

19. Discard the filtrate and reuse the collection tube.

20. Transfer the remaining lysate from Step 15 to the MicroElute® LE DNA Column.

21. Centrifuge at maximum speed for 1 minute.

22. Discard the filtrate and reuse the collection tube.

23. Add 500 μL HBC Buffer. Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

24. Centrifuge at maximum speed for 30 seconds.

25. Discard the filtrate and collection tube.

26. Insert the MicroElute® LE DNA Column into a new 2 mL Collection Tube.

27. Add 700 μL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions.

28. Centrifuge at maximum speed for 30 seconds.

29. Discard the filtrate and reuse the collection tube

30. Repeat Steps 27-29 for a second DNA Wash Buffer wash step.

31. Centrifuge the empty MicroElute® LE DNA Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

32. Transfer the MicroElute® LE DNA Column into a nuclease-free 1.5 mL microcentrifuge tube (not provided).

33. Add 15-100 μL Elution Buffer heated to 70°C.

34. Let sit at room temperature for 2 minutes.

35. Centrifuge at maximum speed for 1 minute.

36. Repeat Steps 33-35 for a second elution step.

Note: Each 200 μL elution will typically yield of 60-70% of the DNA bound to the column. Two elutions will generally yield ~90%. However, increasing the elution volume will reduce the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 50-100 μL Elution Buffer which slightly reduces overall DNA yield. Volumes lower than 50 μL greatly reduce yields. In some instances yields may be increased by incubating the column at 70°C (rather than room temperature) upon the addition of Elution Buffer.

37. Store eluted DNA at -20°C.

Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at 1-800-832-8896.

Problem	Cause	Solution
A260/A230 ratio is low	Salt contamination	• Repeat the DNA isolation with a new sample. • Perform a second wash with HBC Buffer.
A260/A280 ratio is high	RNA contamination	The protocol does not remove RNA. If desired, add 5 µL RNase A (25 mg/mL) after lysate is cleared and before binding buffers are added. Let sit at room temperature for 5 minutes
Low DNA Yield or no DNA Yield	Poor homogenization of sample	Repeat the DNA isolation with a new sample, be sure to mix the sample with SLX-Mlus Buffer thoroughly. Use a commercial homogenizer if possible.
	DNA washed off	Make sure HBC Buffer is mixed with isopropanol and DNA Wash Buffer is mixed with ethanol.
Problems in downstream applications	BSA not added to PCR mixture	Add BSA to a final concentration of 0.1 μg/mL to the PCR mixture.
	Too much DNA inhibits PCR reactions	Dilute the DNA elute used in the downstream application if possible.
	Non-specific bands in downstream PCR	Use hot-start Taq polymerase mixture.
Problems in downstream applications	Inhibitory substance in the eluted DNA	Check the A260/A230 ratio. Dilute the elute to 1:50 if necessary

 

Overview

E.Z.N.A.® FFPE DNA Kit combines MicroElute® LE DNA Column technology with a proprietary buffer system to provide a fast and easy method for DNA isolation from FFPE samples. The sample is heat-treated with FTL2 Buffer followed by Proteinase K digestion to release DNA. After adjusting the binding conditions with ethanol, the lysate is applied to the MicroElute® LE DNA Column to bind DNA. Cellular debris and proteins are effectively removed during the wash steps. High-quality DNA is eluted in sterile deionized water or low salt buffer.

Binding Capacity: Each MicroElute® LE DNA Column can bind approximately 100 µg DNA. Using greater than 30 mg FFPE tissue is not recommended.

New in this Edition:

January 2019

• Omega Bio-tek has a new logo.

February 2017

• FTL Buffer has been replaced with FTL2 Buffer.

December 2013

• The MicroElute® DNA Mini Columns have been replaced with MicroElute® LE DNA Columns. The redesign increases DNA recovery by reducing the HiBind® matrix retention volume.

June 2013

• HB Buffer has been replaced by HBC Buffer. Isopropanol is required and supplied by the user.

• Equilibration Buffer is no longer included with this kit. An optional Column Equilibration Protocol has been added to the protocol for your convenience.

• Equilibration Buffer is replaced with 3M NaOH provided by the user.

Kit Contents

Product	D3399-00	D3399-01
Number of Purifications	5 preps	50 preps
MicroElute® LE DNA Columns	5	50
2 mL Collection Tubes	15	150
BL Buffer	1.5 mL	12 mL
FTL2 Buffer	1.5 mL	12 mL
HBC Buffer	5 mL	25 mL
DNA Wash Buffer	2.5 mL	25 mL
Elution Buffer	2 mL	30 mL
Proteinase K Solution	150 µL	1.5 mL
User Manual

 

Storage and Stability

All components of the E.Z.N.A.® FFPE DNA Kit can be stored at room temperature and are guaranteed for at least 12 months from the date of purchase. Proteinase K Solution can be stored at room temperature for 12 months. For long-term storage (>12 months), store Proteinase K Solution at 2-8°C. Under cool ambient conditions, a precipitate may form in the BL Buffer. In case of such an event, heat the bottle at 37°C to dissolve. Store BL Buffer at room temperature.

Preparing Reagents

1. Dilute HBC Buffer with 100% isopropanol as follows and store at room temperature.

Kit	100% Isopropanol to be Added
D3399-00	2 mL
D3399-01	10 mL

2. Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.

Kit	100% Ethanol to be Added
D3399-00	10 mL
D3399-01	100 mL

E.Z.N.A.®FFPE DNA Kit Protocols

E.Z.N.A.®FFPE DNA Kit Protocol - Xylene Extraction Method

Materials and Equipment to be Supplied by User:

• 100% ethanol

• 100% isopropanol

• Xylene

• Microcentrifuge capable of 14,000 x g

• Vortexer

• 1.5 mL or 2 mL nuclease-free microcentrifuge tubes

• Nuclease-free pipette tips

• Water baths or heat blocks capable of 37°C, 55°C, 70°C, and 90°C

• Optional: RNase A, 20 mg/mL • Optional: 3M NaOH

Before Starting:

• Heat the water bath or heat block to 37°C

• Heat the water bath or heat block to 55°C

• Heat the water bath or heat block to 90°C

• Heat Elution Buffer to 70°C for the elution step

• Prepare the HBC Buffer and DNA Wash Buffer according to the instructions in the Preparing Reagents section on Page 4

1. Add 1 mL xylene to a 1.5 mL or 2 mL microcentrifuge tube (not provided).

2. Cut 3-8 paraffin sections 5-10 µm thick. Note: Do not use the first 2-3 sections.

3. Immediately place the section(s) into the tube containing xylene.

4. Vortex for 20 seconds to mix thoroughly.

5. Centrifuge at maximum speed (≥13,000 x g) for 2 minutes at room temperature.

6. Aspirate and discard the supernatant. Do not disturb the pellet.

7. Add 1 mL 100% ethanol. Vortex to mix thoroughly.

8. Centrifuge at maximum speed for 2 minutes at room temperature.

9. Aspirate and discard the supernatant. Do not disturb the pellet.

10. With the lid open, dry the pellet at 37°C for 15 minutes. Carefully aspirate any residual ethanol with a pipettor before proceeding to the next step.

11. Add 200 µL FTL2 Buffer and pipet up and down to resuspend the pellet.

12. Add 20 µL Proteinase K Solution. Vortex to mix thoroughly.

13. Incubate at 55°C for 3 hours. Note: Incubation can proceed overnight.

14. Incubate at 90°C for 10-30 minutes.

15. Centrifuge the tube briefly to collect any liquid adhering to the lid.

Optional: If RNA-free gDNA is required, add 10 µL RNase A (20 mg/mL, not provided) and let sit for 5 minutes at room temperature.

16. Add 220 μL BL Buffer. Vortex to mix thoroughly.

17. Add 250 μL 100% ethanol. Vortex to mix thoroughly.

18. Insert a MicroElute® LE DNA Column in a 2 mL Collection Tube.

E.Z.N.A.®FFPE DNA Kit Protocols

Optional Protocol for Column Equilibration:

1. Add 100 µL 3M NaOH to the MicroElute® LE DNA Column.

2. Centrifuge at maximum speed for 30-60 seconds.

3. Discard the filtrate and reuse the collection tube.

19. Transfer the entire sample from Step 17 (including any precipitate that may have formed) to the MicroElute® LE DNA Column.

20. Centrifuge at 10,000 x g for 1 minute at room temperature.

21. Discard the filtrate and the collection tube.

22. Transfer the MicroElute® LE DNA Column to a new 2 mL Collection Tube.

23. Add 500 μL HBC Buffer.

Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

24. Centrifuge at 10,000 x g for 1 minute at room temperature.

25. Discard the filtrate and the collection tube.

26. Transfer the MicroElute® LE DNA Column to a new 2 mL Collection Tube.

27. Add 700 μL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions.

28. Centrifuge at 10,000 x g for 1 minute at room temperature.

29. Discard the filtrate and reuse the collection tube.

30. Repeat Steps 27-29 for a second DNA Wash step.

31. Centrifuge the empty MicroElute® LE DNA Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

32. Transfer the MicroElute® LE DNA Column into a new 1.5 mL microcentrifuge tube.

33. Add 50-75 µL Elution Buffer heated to 70°C directly to the center of the column membrane.

34. Let sit for 3 minutes at room temperature.

35. Centrifuge at maximum speed for 1 minute.

36. Repeat Steps 33-35 for a second elution step.

37. Store eluted DNA at -20°C.

E.Z.N.A.®FFPE DNA Kit Protocol - Heat Extraction Method

Materials and Equipment to be Supplied by User:

• 100% ethanol

• 100% isopropanol

• Xylene

• Microcentrifuge capable of 14,000 x g

• Vortexer

• 1.5 mL or 2 mL nuclease-free microcentrifuge tubes

• Nuclease-free pipette tips

• Water baths or heat blocks capable of 55°C, 70°C, and 90°C

• Optional: RNase A, 20 mg/mL

• Optional: 3M NaOH Before Starting:

• Heat the water bath or heat block to 55°C

• Heat the water bath or heat block to 90°C

• Heat Elution Buffer to 70°C for the elution step

• Prepare the HBC Buffer and DNA Wash Buffer according to the instructions in the Preparing Reagents section on Page 4

Note: All centrifugation steps must be performed at room temperature.

1. Add 200 μL FTL2 Buffer into a 1.5 mL or 2 mL microcentrifuge tube (not provided).

2. Cut 3-4 paraffin sections 5-10 µm thick. Note: Do not use the first 2-3 sections.

3. Immediately place the section(s) into the tube containing FTL2 Buffer.

4. Vortex for 20 seconds to mix thoroughly.

5. Incubate at 90°C for 15 minutes to melt the paraffin. Mix the sample a few times by gently shaking the tube 2-3 times. Make sure that the tissue sections stay submerged in the solution.

6. Let sit at room temperature for 5 minutes to allow to cool before adding Proteinase K Solution. Note: If the sample temperature is too high, Proteinase K can be inactivated.

7. Add 20 µL Proteinase K Solution. Vortex to mix thoroughly.

8. Incubate at 55°C for 3 hours.

Note: Incubation can proceed overnight.

9. Centrifuge the tube briefly to collect any liquid adhering to the lid.

Optional: If RNA-free gDNA is required, add 10 µL RNase A (20 mg/mL, not provided) and incubate for 5 minutes at room temperature.

10. Add 220 μL BL Buffer. Vortex to mix thoroughly.

11. Add 250 μL 100% ethanol. Vortex to mix thoroughly.

12. Insert a MicroElute® LE DNA Column in a 2 mL Collection Tube.

Optional Protocol for Column Equilibration:

1. Add 100 µL 3M NaOH to the MicroElute® LE DNA Column.

2. Centrifuge at maximum speed for 30-60 seconds.

3. Discard the filtrate and reuse the collection tube.

13. Transfer the entire sample from Step 11 (including any precipitate that may have formed) to the MicroElute® LE DNA Column.

14. Centrifuge at 10,000 x g for 1 minute at room temperature.

15. Discard the filtrate and the collection tube.

16. Transfer the MicroElute® LE DNA Column to a new 2 mL Collection Tube.

17. Add 500 μL HBC Buffer.

Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

18. Centrifuge at 10,000 x g for 1 minute at room temperature.

19. Discard the filtrate and the collection tube.

20. Transfer the MicroElute® LE DNA Column to a new 2 mL Collection Tube.

21. Add 700 μL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions.

22. Centrifuge at 10,000 x g for 1 minute at room temperature.

23. Discard the filtrate and reuse the collection tube.

24. Repeat Steps 21-23 for a second DNA Wash step.

25. Centrifuge the empty MicroElute® LE DNA Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

26. Transfer the MicroElute® LE DNA Column into a new 1.5 mL microcentrifuge tube.

27. Add 50-75 µL Elution Buffer heated to 70°C directly to the center of the column membrane.

28. Let sit for 3 minutes at room temperature.

29. Centrifuge at maximum speed for 1 minute to elute DNA.

30. Repeat Steps 27-29 for a second elution step.

31. Store eluted DNA at -20°C.

E.Z.N.A.®FFPE DNA Kit Protocol - Vacuum Method

Materials and Equipment to be Supplied by User:

• Vacuum manifold

• Vacuum source or pump

The vacuum method outlined here is an alternative to centrifugation steps presented in the protocols above. Either the xylene or heat extraction method can be used to remove the paraffin prior to DNA extraction via the vacuum method. Carry out deparaffinization, Proteinase K digestion, and column equilibration as indicated in either of the two preceding protocols. Instead of continuing with the initial sample transfer to the MicroElute® LE DNA Column (Step 19 in the Xylene Method, Page 7 or Step 13 in the Heat Method, Page 10), follow the steps below.

Note: Please read through the preceding protocols of this manual before using this protocol.

1. Prepare the vacuum manifold according to manufacturer’ s instructions.

2. Connect the MicroElute® LE DNA Column to the vacuum manifold.

3. Load the sample (from Step 17 in the Xylene Method, Page 6 or Step 11 in the Heat Method, Page 10) onto MicroElute® LE DNA Column.

4. Turn on vacuum source to draw the sample through the column.

5. Turn off the vacuum.

6. Add 500 μL HBC Buffer.

Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 4 for instructions.

7. Turn on vacuum source to draw the HBC Buffer through the column.

8. Turn off the vacuum.

9. Add 700 µL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol before use. Please see Page 4 for instructions.

10. Turn on vacuum source to draw the DNA Wash Buffer through the column.

11. Turn off the vacuum.

12. Repeat Steps 9-11 for a second DNA Wash step.

13. Remove the column from the vacuum manifold and transfer to a new 2 mL Collection Tube.

14. Centrifuge at full speed for 2 minutes to completely dry the membrane.

Note: It is important to dry the column membrane before elution. Residual ethanol may interfere with downstream applications.

15. Place the MicroElute® LE DNA Column into a new 1.5 mL microcentrifuge tube.

16. Add 50-75 µL Elution Buffer heated to 70°C directly to the center of the column membrane.

17. Let sit for 3 minutes at room temperature.

18. Centrifuge at maximum speed for 1 minute to elute DNA.

19. Repeat Steps 16-18 for a second elution step.

20. Store eluted DNA at -20°C.

Troubleshooting Guide

Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at (800-832-8896).

Problem	Cause	Solution
	Incomplete cell lysis or crosslinking removal	Increase incubation time with FTL2 Buffer and protease. Ensure that no visible pieces of tissue remain
	Samples are rich in protein	After applying to column, wash with 300 μL of a 1:1 mixture of BL Buffer and ethanol and then with DNA Wash Buffer.
Problem	Cause	Solution
No DNA eluted	Poor cell lysis due to improper mixing with BL Buffer	Mix thoroughly with BL Buffer prior to loading the MicroElute® LE DNA Column.
	Poor cell and/or protein lysis in FTL2 Buffer	Tissue sample must be cut or minced into small pieces. Increase incubation time at 55°C with FTL2 Buffer to ensure that tissue is completely lysed.
	Ethanol not added to DNA Wash Buffer	Dilute DNA Wash Buffer with the indicated volume of 100% ethanol before use.
	Isopropanol not added to HBC Buffer	Dilute HBC Wash Buffer with the indicated volume of 100% isopropanol before use.
Problem	Cause	Solution
Washing leaves colored residue in column	Incomplete lysis due to improper mixing with BL Buffer	BL Buffer is viscous and the sample must be vortexed thoroughly
	Ethanol not added to DNA Wash Buffer	Dilute DNA Wash Buffer with the indicated volume of 100% ethanol before use.

  

 

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