User Manual

FavorPrep Tissue Genomic DNA Extraction Mini Kit

-- For extraction genomic DNA from animal cells, animal tissues, blood, bacteria,

paraffin fixed tissue, yeast and fungi

Kit Contents:

For Research Use Only

Cat. No:

FATG1 Buffer 1.5 ml 15 ml 30 ml 70 ml

FATG2 Buffer 1.5 ml 15 ml 30 ml 70 ml

W 1 Buffer * (contentrate) 1.3 ml 22 ml 44 ml 124 ml

Wash Buffer ** (concentrate) 1 ml 10 ml 20 ml 55 ml

Elution uffer 1 ml 15 ml 30 ml 90 ml

FATG Mini Column 4 pcs 50 pcs 100 pcs 300 pcs

Collection Tube 8 pcs 100 pcs 200 pcs 600 pcs

Elution Tube 4 pcs 50 pcs 100 pcs 300 pcs

Micropestle 4 pcs 50 pcs 100 pcs 300 pcs

User Manual 1 1 1 1

Preparation of Proteinase K solution (10 mg/ml) by adding ddH

2

O

▪ ddH

2

O volume for Proteinase K 0.1 ml 1.1 ml

Preparation of W 1 Buffer and Wash Buffer by adding ethanol (96 ~ 100%)

* Ethanol volume for W 1 Buffer 0.5 ml 8 ml 16 ml

**Ethanol volume for Wash Buffer 4 ml 40 ml 80 ml

45 ml

210 ml

Specification:

Principle: mini spin column (silica matrix)

Operation time: 30 ~ 60 minutes

Binding capacity: up to 60 µg DNA/ column

Typical yield: 15 ~35 µg/ prep

Column applicability: centrifugation and vaccum

Minimum elution volume: 50 µl

Sample size: < 25 mg animal tissue

1.2 cm mouse tail

Brief procedure:

Sample

Grind the sample

Cell lysis ( FATG1 )

Protein degradation ( Proteinase K )

Cell lysis ( FATG2 )

Important Notes:

1. Buffers provided in this system contain irritants. Wear

gloves and lab coat when handling these buffers.

2. Add 1.1 ml sterile ddH

2

O to Proteinase K tube to

make a 10 mg/ml stock solution. Vortex and make

sure that Proteinase K has been completely

dissolved. Store the stock solution at 4 °C.

3. Add ethanol (96- 100 %) to W1 Buffer and Wash Buffer

when first open.

4. Prepare dry baths or water baths before the

operation: one to 60 °C for step 4 and the other to

70 °C for step 7.

5. Preheat the Elution Buffer to 70 °C for step 13.

6. All centrifuge steps are done at full speed

(~ 18,000 x g) in a microcentrifuge.

centrifuge centrifuge centrifuge

Binding

Washing ( W1 Buffer )

( Wash Buffer )

Elution ( Elution Buffer )

Pure genomic DNA

1 v 1115

Protocol: Isolation of DNA from Animal Tissue

Please Read Important Notes Before Starting Following Steps.

Additional requirment: RNase A (optional), 96~100% ethanol

Hint: Set dry or water baths: 60 °C for step 4 and 70 °C for step 6.

1. Cut up to 25 mg tissue sample to a microcentrifuge tube (not provided). Use provided Micropestle to grind

the tissue sample. Or you can grind the tissue sample in liquid nitrogen with mortar and pestle then transfer the

powder to a microcentrifuge tube.

--- If DNA is prepared from spleen tissue, no more than 10 mg should be used.

2. Add 200 µl FATG1 Buffer and mix well by Micropestle or pipette tip.

3. Add 20 µl Proteinase K (10mg/ml) to the sample mixture. Mix thoroughly by vortexing.

4. Incubate at 60 °C until the tissue is lysed completely (1~3 h). Vrotex occasionally during incubation.

--- Sample can be incubated overnight as well for complete lysis.

5. (Optional) If RNA-free genomic DNA is required, add 4 µl of 100 mg/ml RNase A (not provided). Mix thoroughly

by vortexing and incubate at room temperature for 2 min.

6. Add 200 µl FATG2 Buffer to the sample mixture, mix thoroughly by pulse-vortexing and incubate at 70 °C for 10 min.

7. Add 200 µl ethanol (96-100%) to the sample mixture. Mix thoroughly by pulse-vortexing.

8. Briefly spin the tube to remove drops from the inside of the lid.

9. Place a FATG Mini Column in a Collection Tube. Transfer the mixture (including any precipitate) carefully to the

FATG Mini Column. Centrifuge at full speed (~18,000 x g) for 1 min then place the FATG Mini Column to a new

Collection Tube.

10. Add 400 µl W1 Buffer to the FATG Mini Column. Centrifuge at full speed for 1 min then discard flow-through.

---Make sure that ethanol has been added into W1 Buffer when first open.

11. Add 750 µl Wash Buffer to the FATG Mini Column. Centrifuge at full speed for 1 min then discard flow-through.

---Make sure that ethanol has been added into Wash Buffer when first open.

12. Centrifuge at full speed for an additional 3 min to dry the column.

--- Important Step! This step will remove the residual liquid.

13. Add 100 µl of preheated Elution Buffer or ddH

2

O (pH 7.5-9.0) to the membrane of the FATG Mini Column.

Stand the FATG Mini Column for 3 min.

--- Important Step! For effective elution, make sure that the elution solution is dispensed onto the membrane

center and is absorbed completely.

--- If less sample to be used, reduce the elution volume to 50 µl to increase DNA concentration and do not elute

the DNA using less than suggested volume (50 µl). It will lower the final yield.

14. Centrifuge at full speed for 2 min to elute DNA.

Protocol: Isolation of DNA from Animal Cultured Cells

Please Read Important Notes Before Starting Following Steps.

Additional requirment: RNase A (optional), 96~100% ethanol, trypsine or cell scraper (for monolayer cell ), PBS

Hint: Set dry or water baths: 60 °C and 70 °C

1. Harvest cells

a. Cells grown in suspension

і. Transfer the appropriate number of cell ( up to 1 x 10 ) to a microcentrifuge tube.

іі. Centrifuge at 300 x g for 5 min. Discard supernatant carefully and completely.

b. Cells grown in monolayer

і. Detach cells from the dish or flask by trypsinization or using a cell scraper. Transfer the appropriate

7 number of cell ( up to 1 x 10 ) to a microcentrifuge tube.

iі. Centrifuge at 300 x g for 5 min. Discard supernatant carefully and completely.

2. Resuspend cell pellet in PBS to a final volume of 200 µl.

3. Follow the Animal Tissuel Protocol starting from step 2.

Protocol: Isolation of Genomic DNA and Viral DNA from Blood

Please Read Important Notes Before Starting Following Steps.

Additional requirment: RNase A (optional), 96~100% ethanol, PBS

Hint: Set dry or water baths: 60 °C for step 3 and 70 °C for step 4.

1. Transfer up to 200 µl sample ( whole blood, serum, plasma, body fluids, buffy coat) to a microcentrifuge tube.

--- If the sample volume is less than 200 µl , add the appropriate volume of PBS.

2. (Optional) If RNA-free genomic DNA is required, add 4 µl of 100 mg/ml RNase A (not provided). Mix thoroughly

by vortexing and incubate at room temperature for 2 min.

3. Add 20 µl Proteinase K to the sample, and then add 200 µl FATG2 Buffer to the sample. Mix thoroughly by

pulse-vortexing. Incubate at 60 °C for 30 min. Vrotex occasionally during incubation.

4. Incubate at 70 °C for 10 min.

2. Follow the Animal Tissuel Protocol starting from step 7.

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Protocol: Isolation DNA from Bacteria

Please Read Important Notes Before Starting Following Steps

Additional equipment: • RNase A (optional), 96~100% ethanol,

• For Gram-positive bacteria: lysozyme reaction solution (20 mg/ml lysozyme;

20 mM Tris-HCI, pH 8.0; 2mM EDTA; 1.2 % Triton)

Hint: Set dry or water baths: 60 °C for step 4 and 70 °C for step 6.

l. For bacterial cultures

1. Transfer 1 ml well-grown bacterial culture to a microcentrifuge tube (not provided).

2. Descend the cells by centrifuging at full speed for 2 min and discard supernatant completely.

3. Follow the Animal Tissue Protocol starting from step 2.

ll. For bacterial in biological fluids

1. Collect cells by centrifuging biological fluids at 7,500 rpm (5,000 x g) for 10 min and discard supernatant

completely.

2. Follow the Animal Tissue Protocol starting from step 2. lll. For bacteria from eye, nasal, pharyngeal, or other swabs

1. Soak the swabs in 2 ml PBS at room temperature for 2- 3 hr.

2. Collect cells by centrifuging at 7,500 rpm (5,000 x g) for 10 min and discard supernatant completely.

3. Follow the Animal Tissue Protocol starting from step 2.

lV.For Gram-positive bacteria

HINT: Set dry or water baths: one to 37 °C, another to 60 °C and the other to 95 °C.

1. Transfer 1 ml well-grown bacterial culture to a microcentrifuge tube (not provided).

2. Descend the cells by centrifuging at full speed for 2 min and discard supernatant completely.

3. Resuspend the cell pellet in 200 µl lysozyme reaction solution (20 mg/ml lysozyme; 20 mM Tris-HCI, pH 8.0;

2mM EDTA; 1.2 % Triton). Incubate at 37 °C for 30~60 min.

4. (Optional) If RNA-free genomic DNA is required, add 4 µl of 100 mg/ml RNase A (not provided).

Mix thoroughly by vortexing and incubate at room temperature for 2 min.

5. Add 20 µl Proteinase K to the sample, and then add 200 µl FATG2 Buffer to the sample. Mix thoroughly

by pulse-vortexing. Incubate at 60 °C for 30 min and vrotex occasionally during incubation.

6. Do a furture incubation at 95 °C. for 15 min.

7. Follow the Animal Tissue Protocol starting from step 7.

Protocol: Isolation of DNA from Yeast

Please Read Important Notes Before Starting Following Steps.

Additional equipment: • RNase A (optional), 96~100% ethanol,

• Zymolase or lyticase, 200 U for one preparation

• Sorbitol buffer (1M sorbitol; 100 mM EDTA; 14 mM ß-mercaptoethanol)

HINT: Set dry or water baths: one to 30 °C, another to 60 °C and the other to 70 °C.

1. Transfer 3 ml log-phase (OD600 = 10) yeast culture to a microcentrifuge tube (not provided).

2. Descend the cells by centrifuging at 7,500 rpm (5,000 x g) for 10 min. Discard supernatant completely.

3. Resuspend the cell pellet in 600 µl sorbitol buffer (1M sorbitol; 100 mM EDTA; 14 mM ß-mercaptoethanol).

Add 200 U zymolase or lyticase and incubate at 30 °C for 30 min.

4. Centrifuge at 7,500 rpm (5,000 x g) for 5 min. Remove supernatant by pipetting.

5. Follow the Animal Tissue Protocol starting from step 2.

Protocol: Isolation of DNA from Dried Blood Spot

Please Read Important Notes Before Starting Following Steps.

Additional equipment: • RNase A (optional), 96~100% ethanol

HINT: HINT: Set dry or water baths: one to 85 °C, another to 60 °C and the other to 70 °C.

1. Cut the filter paper (e.g. S&S903) with dried blood spot into a microcentrifuge tube. Add 200 µl FATG1

Buffer and incubate at 85 °C for 10 min.

2. Add 20 µl Proteinase K to the sample mixture. Mix thoroughly by vortexing. Incubate at 60 °C for 1 hr.

Vrotex occasionally during incubation.

3. Follow the Animal Tissue Protocol starting from step 6.

Protocol: Isolation of DNA from Fixed Tissue

Please Read Important Notes Before Starting Following Steps.

Additional equipment: • RNase A (optional), 96~100% ethanol,

• Xylene - for paraffin-embedded tissues

Hint: Set dry or water baths: 60 °C and 70 °C

I. For paraffin-embedded tissues

1. Cut up to 25 mg paraffin-embedded tissue sample to a microcentrifuge tube (not provided).

2. Add 1 ml xylene, mix well and incubate at room temperature for 30 min.

3. Centrifuge at full speed for 5 min. Remove supernatant by pipetting.

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4. Add 1 ml ethanol (96- 100 %) to the deparaffined tissue, mix gently by vortexing.

5. Centrifuge at full speed for 3 min. Remove supernatant by pipetting.

6. Repeat step 4 and 5.

7. Incubate at 37 °C for 10 ~15 min to evaporate ethanol residue completely.

8. Grind the tissue sample by micropestle or liquid nitrogen and follow the Animal Tissue Protocol starting

from step 2.

ll. For formalin-fixed tissues

1. Wash 25 mg tissue sample twice with 1 ml PBS to remove formalin.

2. Grind the tissue sample by micropestle or liquid nitrogen and follow the Animal Tissue Protocol starting

from step 2.

Troubleshooting

Problem/ Possible reasons

• Low or no yield of genomic DNA

Low amount of cells in the sample

To much amount of sample was used

Poor cell lysis

Solutions

Increase the sample size or concentrate a larger sample volume to 200 µl.

Reduce the sample volume.

Use a fresh or well-stored Proteinase K stock solution.

Do not add Proteinase K into FATG2 Buffer directly.

Poor cell lysis because of insufficient Extend incubation time and make sure that no residual particle remain.

Insufficient binding of DNA to column’s membrane

Incorrect preparation of Wash Buffer

Elution of genomic DNA is not efficient pH of water (ddH

2

O) for elution is

Elution Buffer or ddH

2

O is not

Make sure the pH of ddH

2

O is between 7.5-9.0.

Use Elution Buffer (provided) for elution .

before centrifugation.

2

O is added, stand the FATG Column for 5 min

• Column is clogged

Lysate contains insoluble residues

Sample is too viscous

Insufficient activity of Proteinase K

Remove insoluble residues (e.g. bone or hair) by centrifugation.

Reduce the sample volume.

• Poor quality of genomic DNA

A260/A280 ratio of eluted DNA is low

Use a fresh or well-stored Proteinase K stock solution.

Do not add Proteinase K into FATG2 Buffer directly.

vortexing.

remain.

mixing with FATG2 buffer incubation time

A260/A280 ratio of eluted DNA is high

A lot of residual RNA in eluted DNA lysate before added RNase A

Degradation of eluted DNA

Sample is old

Follow the Animal Tissue Protocol step 5 to remove RNA.

adding FATG2 Buffer when using optional RNase A step.

Always use fresh or well-stored sample for genomic DNA extraction.

Use fresh running buffer for gel electrophoresis.

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