EPOCH LIFE SCIENCE 2460050 GenCatch Blood and Tissue Genomic Mini Prep Kit User Guide

GenCatch™ Blood & Tissue
Genomic Mini Prep Kit
User’s Guide for
Genomic DNA Purification

2460050 GenCatch Blood and Tissue Genomic Mini Prep Kit

For Research Use Only

Quick Start Procedure

This Quick Start Procedure Is For Experienced Users Only.
First time users are strongly recommended to read through the detailed protocol in section 4.
Before you start:
Add 60 ml (50 preps) or 180 ml (250 preps) 98-100% ethanol to WS Buffer.

Overview

GenCatch™ plus Genomic DNA Extraction Kit provides a fast and efficient method to purify genomic DNA from various sources such as cultured cells, animal tissues, whole blood, buffy coat, lymphocytes, plasma, serum, bacteria, yeasts, DNA virus, paraffin-embedded tissues, etc. without the need of time- consuming phenol/chloroform extraction and ethanol precipitation. This simple, convenient spin-column format can isolate genomic DNA of predominantly 20-30 kb free of protein and salt contaminants. 0.3 ml blood volume will yield up to 15ug of genomic DNA.
Preparation time: 1-4 hr depends on sample sources
Downstream Applications:

• Southern blotting
• RAPD, RFLP
• Restriction enzyme digestion
• Genomic library construction
• PCR, Real-Time PCR
• Genotyping

Product Contents

GenCatch™ Plus Blood/Tissue Genomic DNA Kit contains sufficient reagents for 50 (Cat. No. 2460050) and 250 (Cat. No. 2460250) genomic DNA purifications respectively.

Add 60 ml 150 preps) or 180 ml 1250 preps) 98-100% ethanol to WS buffer bottle when first opened.
Add 1 ml sterile ddH2O to reconstitute one tube of the provided Proteinase K by overtaxing for 1 minute. Make sure that proteinase K has been completely dissolved. The solution should appear clear. The concentration of the proteinase K stock is 10 mg/ml. Store the solution at 4°C.
Storage Conditions (except proteinase K):
Store at room temperature
All components are guaranteed for 24 months from the date of purchase, when stored under specified conditions and used as described in this manual. GenCatch™ Mini columns should be kept sealed in the zip lock bag provided during storage and away from any heating source.

Protocol

First time users are strongly recommended to read through this detailed protocol instruction.
For technical support please contact us at [email protected]
Before you start:
Add 60 ml (50 preps) or 180 ml (250 preps) 98-100% ethanol to Buffer WS before use (see instructions on bottle label).
i. Blood Protocol:
For samples including whole blood (anti-coagulant added.)

1. Pipet up to 300 µl sample into a 1.5 ml sterile Eppendorf tube. Use PBS to make up to 200 µl if volume is less than 200 µl. Add 1.0 ml RL Buffer, mix well by inverting, incubate for 5 minutes to lyse red blood cell.

2. Centrifuge at 5,000 rpm for 3 minutes, discard the supernatant.

3. Add 0.8 ml RL Buffer and invert several times to mix well. Centrifuge at 5000 rpm for 2 minutes, discard the supernatant.

4. Add 20 µl Proteinase K and 180 µl LYS Buffer into the sample. Mix immediately by vortexing for 20 seconds. Do not add and keep proteinase K directly in LYS Buffer. When sample volume ≥200 µl, increase the amount of proteinase K and LYS Buffer proportionally.

5. Incubate at 60°C for 30 minutes to lyse the sample. Vortex or invert the sample every 3-5 minutes during incubation.
   Ensure complete sample lysis, sample should appear translucent; buffy coat should not contain insoluble residues.
   If RNA-free genomic DNA is desired, add 10 µl of 50 mg/ml RNase A to the sample for at least 10 minutes at room temperature at this step.

6. Add 300 µl FX Buffer into the sample, and mix well by inverting.

7. Follow the wash step on page 11.

ii. Tissue Protocol:

1. -a Cut 30 mg of tissue (15 mg spleen) into small pieces and place the sample into a 1.5 ml sterile Eppendorf tube. Add 180 µl LYS Buffer to homogenize the sample. If the sample size is larger than 30 mg, Increase the amount of LYS Buffer proportionally.
   For direct blood (nucleated blood cell, like bird or fish), apply up to 20 µl nucleated blood cell sample into a 1.5 ml sterile Eppendorf tube, then add 180 µl LYS Buffer and mix well.

2. Add 20 µl Proteinase K to the sample. Mix immediately by vortexing for 20 seconds. If RNA-free genomic DNA is desired, add 10 µl of 50 mg/ml RNase A to the sample.

3. Incubate at 60°C for 1 hour to lyse the sample. If tissue is difficult to lyse, increase the incubation time to 2-3 hours. Vortex or invert mix the sample every 10-15 minutes
   during incubation. Ensure complete sample lysis, sample should appear translucent.

4. Add 300 µl FX Buffer into the sample, and mix well by inverting.

5. Follow the wash step on page 11.

iii. Mouse Tail Protocol:

1. Cut into small pieces of a segment of mouse tail of up to 0.5 cm. Place the sample into a 1.5 ml sterile Eppendorf tube.
   Segment close to the tail tip is preferred. Segment away from the tip is thicker and takes longer time to lyse completely.

2. Add 20 µl proteinase K and 200 µl LYS Buffer to the sample. Mix immediately by vortexing for 20 seconds. If RNA-free genomic DNA is desired, add 10 µl of 50 mg/ml RNase A to the sample.

3. Incubate at 60°C for 1-4 hours or overnight to lyse the tail tissue. Vortex or invert mix the sample every 20-30 minutes during incubation. Ensure complete sample lysis, ample should appear translucent.

4. Add 300 µl FX Buffer into the sample, and mix well by inverting.

5. Follow the wash step on page 11.

iv. Serum Protocol:
For samples including buffy coat, serum, plasma, body fluid, 10 lymphocytes and cultured cells in 200 µl PBS. Sample volume should not exceed 200ul.

1. Add 20 µl proteinase K and 150 µl FX Buffer to the sample. Mix immediately by vortexing for 20 seconds If RNA-free genomic DNA is desired, add 10 µl of 50 mg/ml RNase A to the sample.
2. Incubate at 60°C for 40 minutes to lyse the sample. Vortex or invert mix the sample every 5 minutes during incubation. Ensure complete sample lysis, sample should pear translucent.
3. Add 150 µl FX Buffer into the sample, mix well by inverting
4. Follow the wash step on page 11.

v. Paraffin-Embedded Tissue Protocol:

1. Cut a small section of paraffin-embedded tissue (about 25 mg) and put the sample into a 1.5 ml. sterile Eppendorf tube.
2. Add 1 ml xylene and incubate at room temperature with occasional mixing for 30 minutes to extract paraffin from tissue.
3. Centrifuge at full speed for 5 minutes. Remove the supernatant by pipetting.
4. Add 1 ml of absolute ethanol to the tissue pellet, mix and centrifuge at full speed for 5 minutes. Remove ethanol-containing xylene residue by pipetting.
5. Evaporate ethanol residue by incubating at 37°C for 10 minutes.
6. Resuspend the pellet in 180 pl LYS Buffer.
7. Add 20 pl proteinase K to the sample. Mix immediately by vortexing for 20 seconds. If RNA-free genomic DNA is desired, add 10 pl. of 50 mg/ml RNase A to the sample.
8. Incubate at 60°C for 1 hour to lyse the sample. If tissue is difficult to lyse, increase the incubation time to 2-3 hours. Vortex or invert mix the sample every 10-15 minutes during incubation. Ensure complete sample lysis, sample should appear translucent.
9. Add 300 µl FX Buffer into the sample, and mix well by invert.
10. Follow the wash step on page 11.

vi. Bacteria Protocol:
For Bacteria (Gram-positive or negative)
a. Pellet log-phase grown bacteria of up to 109 cells (or up to 3 ml culture) by centrifugation at 7500 rpm for 10 minutes.
b. Resuspend the pellet in 200 pl lysozyme reaction solution (20 mM Tris-HCI, pH 8.0; 2 mM EDTA; 20 mg/ml lysozyme). Incubate at 37°C for 30minutes.
c. Add 20 pl. Proteinase K and 200 pl FX Buffer to the sample. Mix immediately by vortexing for 20 seconds. If RNA-free genomic DNA is desired, add 10 pl. of 50 mg/ml RNase A to the sample.
For Bacteria in biological fluids
a. Pellet cells by centrifugation at 7500 rpm for 10 minutes.
b. Resuspend the pellet in 180 µl LYS Buffer.
For Bacteria from eye, nasal or pharyngeal swabs
a. Collect bacterial cells by rinsing and soaking the swabs in 2 ml PBS at room temperature for 2-3 hours.
b. Pellet cells by centrifugation at 7500 rpm for 10 minutes.
c. Resuspend the pellet in 180 µl LYS Buffer.

1. Incubate at 60°C for 30 minutes to lyse the bacterial cells. Vortex or invert mix the sample every 5 minutes during incubation.
   Ensure complete sample lysis, sample should appear translucent.

2. Add 150 µl FX Buffer into the sample, and mix well by invert.

3. Follow the wash step on page 11.

vii. Yeast Protocol:

1. Pellet log-phase grown yeast cells of up to 108 (or up to 3 ml culture) at 7500 rpm for 10 minutes.
2. Resuspend the pellet in 500 µl sorbitol reaction solution (1 M sorbitol; 100 mM EDTA; 14 mM (3-mercaptoethanol; 200 U lyticase or zymolase). If RNA-free genomic DNA is desired, add 10 µl. of 50 mg/ml RNase A to the sample.
3. Incubate at 30°C for 30 minutes.
4. Pellet cells by centrifugation at 7500 rpm for 5 minutes. Resuspend the pellet in 180 pl. LYS Buffer.
5. Add 20 µl. proteinase K and 200 µl. LYS Buffer to the sample. Mix immediately by vortexing for 20 seconds. If RNA-free genomic DNA is desired, add 10 µl. of 50 mg/ml RNase A to the sample.
6. Incubate at 60°C for 1-4 hours or overnight to lyse the tail. tissue. Vortex or invert mix the sample every 20-30 minutes during incubation. Ensure complete sample lysis, sample should appear translucent.
7. Add 300 µl FX Buffer into the sample, and mix well by invert.
8. Follow the wash step on page 11.

viii. Virus Protocol:

1. To prepare viral DNA from blood or body fluid, the Blood Protocol is recommended.
2. To prepare integrated viral DNA, Tissue Protocol is recommended.

ix. DNA Wash Protocol

1. Adjust the temperature to 70°C and incubate for another 20 minutes.
2. Preheat 10 mM Tris-HCl (pH 9.0), ddH2O or TE buffer at 70°C (500 µl/prep) for DNA elution.
3. Add 200 µl of absolute ethanol or isopropanol to the sample and mix by vortexing. If the sample is more than 550ul, increase the amount of ethanol proportionally.
4. Place a GenCatch™ Mini column onto a Collection Tube. Transfer all the mixture into the column. Centrifuge at 8000 rpm for 2 minutes. Place the column onto a new Collection Tube.
5. Wash the column twice with 0.5 ml WS Buffer by centrifugation at 8000 rpm for 2 minutes. Discard the flow-through after centrifugation. Make sure ethanol is added to WS Buffer when first opened.
6. Centrifuge the column at full speed for another 2 minutes to remove ethanol residue.
7. Place the column onto a new 1.5 ml tube and elute DNA with 200 µl of the preheated elution solution from Step 8. Elution volume can be adjusted for concentration, but should be more than 20µl.
8. Stand the column for 1-5 minutes, and centrifuge for 1-2 minutes to elute DNA.
9. Store the eluted DNA at -20°C or proceeds directly to the downstream experiment.

Troubleshooting Guide

The following guide addresses some of the most common problems. A database of user raised questions and answers are being build at support.epochbiolabs.com.

Incomplete digestion of hemoglobin
No alcohol or alcohol of incorrect amount is added to the sample before loaded into the column
WS Buffer does not contain ethanol| Vortex the sample after Proteinase K is added. Mix the sample every
3-5 minutes during incubation.
Before passing the column, add 210 µl of absolute alcohol to the sample.
Make sure that ethanol is added into the WS Buffer bottle when first open.
Low or no yield of DNA| Sample contains too low amount of genomic DNA
Blood or cell sample is not lysed completely
No alcohol or alcohol of incorrect amount is added to the sample before loaded into the column
Elution solution is not preheated at 70°C
pH of the elution solution is too low
WS Buffer does not contain ethanol| Increase the sample amount, Proteinase K and buffer proportionally. If the sample is whole blood, prepare buffy coat from a larger volume of blood.
Add another 20 µl fresh Proteinase K per sample and repeat incubation.
Before passing the column, add 210 µl of absolute alcohol to the sample.
Preheat the elution solution at 70°C before used.
Make sure that the pH of 10 mM Tris-HCl, ddH2O or TE buffer for elution is between 8.0-9.0.
Make sure that ethanol is added into the WS Buffer bottle when first open.
Column is clogged when passing the sample| Tissue sample still remains undigested after lysis
Blood sample contains clots
Sample is very viscous| After Proteinase K digestion, centrifuge the sample at full speed for 5 minutes to remove undigested remains.
Use whole blood sample mixed well with anti-coagulant to prevent formation of blood clot.
Do not use blood clot for genomic DNA extraction.
Too much sample is used. Reduce the sample amount.
A260/A280
ratio of eluted genomic DNA is low| Protein in the sample is not completely degraded
Protein in the sample is not completely degraded
No alcohol or alcohol of incorrect amount is added to the sample before loaded into the column
Eluted genomic DNA contains contaminants
Eluted genomic DNA carries ethanol
Using ddH2O of acidic pH
(5.0-6.0) to dilute DNA sample for spectrophotometric analysis| Vortex the sample after Proteinase K is added. Mix the sample at constant intervals during incubation.
Add 20 µl fresh Proteinase K per sample and continue incubation.
Before passing the column, add 210 µl of absolute alcohol to the sample.
Do not touch the rim of the column during sample or buffer loading.
After the final wash, centrifuge the column at full speed for another 2 minutes to remove the ethanol residue completely.
Use 10 mM Tris-HCl of pH 7.5 or TE buffer to dilute the DNA sample.
A260/A280 ratio of eluted genomic DNA is high (  1.9)| Eluted genomic DNA contains a lot of RNA| Add RNase A to the sample as described in the protocol.
Genomic DNA appears smearing and degraded| Sample is not fresh or stored improperly for a long time
Blood sample is not fresh or stored  improperly for a long time
Gel electrophoresis is performed in used running buffer contaminated with DNase
Paraffin-embedded tissue is used as sample| Flash freeze fresh sample in liquid nitrogen and store at -80°C if not used immediately.
Use fresh blood or blood stored at room temperature for fewer than 2 days.
Use fresh TAE or TBE running buffer for electrophoresis.
Genomic DNA isolated from this kind of sample is usually degraded. It is still suitable for PCR application, but is not recommended for Southern blotting and restriction analysis.

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References

• Epoch Life Science
• Epoch Life Science

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