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Genomic DNA Extraction Kit 

This kit is designed for the rapid preparation of genomic DNA from various sources including: cultured cell,tissue,blood etc. It is designed for the rapid preparation of 3-20 μg DNA from various sources.
Cat. No. Size Price Quantity  
TB50004A 50 preps USD 87
TB50004B 200 preps USD 280
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

Contents Volume
Buffer A 12 ml
Buffer B 12 ml
Buffer W1 21 ml
Buffer W2 8 ml
Elution Buffer 12 ml
Proteinase K 1 ml
Spin Column 50 preps
Collection Tube 50 preps

Storage and Stability

2-8°C 12 months

Description

This kit is designed for the rapid preparation of genomic DNA from various sources including: cultured cell,tissue,blood etc. It is designed for the rapid preparation of 3-20 μg DNA from various sources including: 50~200 μl of mammalian whole blood (with anticoagulant), 103 ~106 cultured cells,25 mg of mammalian tissues. The extraction process combines an initial lysis step and a subsequent proteolytic digestion step using proteinase K with a highly efficient process for binding gDNA to the surface of a spin column membrane. High-purity gDNA is eluted from the membrane after washing, which is highly suited for a variety of applications, including restriction analysis, sequencing, PCR, southern blotting etc.

DNA Yield

Sample Amount DNA Yield
Animal tissues 25 mg 5-20 μg
Cultured cells 5×106 5-20 μg
Mammalian blood <200 μl 3-6 μg
Bird, fish or amphibian blood 5-10 μl 5-20 μg

Important notes before starting

  1. For research only. Not intended for human or diagnostic.
  2. RNase A (not provided) can be used if RNA-free DNA is required.
  3. Buffer B and Buffer W1 contain strong protein denaturant. Wear gloves, lab coat, etc.when handling these buffers.
  4. Make sure that no precipitates have formed in Buffer B and Buffer W1. If necessary, warm the solutions and mix well until the solutions become clear again.
  5. All centrifugation steps should be carried out at room temperature (RT, 15-25°C).
  6. Avoid freeze/thaw cycles of samples. Freeze/thaw cycles of blood sample would result in low yield of genomic DNA or DNA length less than 30kb.
  7. Add 7 ml and 32 ml ethanol(96-100%) into Buffer W1 and Buffer W2 respectively when first use.

Procedure

A. DNA Extracted From Tissue

  1. Grind less than 25 mg tissue(up to 10 mg of spleen) in liquid and add 180 μl Buffer A.(Too much sample may results in low yield of genomic DNA,Small pieces enable more efficient lysis.)
  2. Add 20 μl of Proteinase K, mix thoroughly by vortexing, and incubate at 55°C until the tissue is completely lysed.(Incubating time may varied depend on the type of tissue processed, usually in 1-3 hours and overnight for mouse tail)
  3. If RNA-free genomic DNA is required, add 4 μl RNaseA (100 mg/ml) and mix the sample by inverting the tube 2–5 times. Incubate the mixture at RT for 2 minutes. (Optional)
  4. Add 200 μl Buffer B to the sample, mix by vortexing, and incubate at 70°C for 10 min to ensure the sample lysed is clear and homogeneous.(White precipitates may form when Buffer B is added. They will not interfere with the procedure and will dissolve during the incubation at 70°C)
  5. Add 200 μl ethanol (96-100%), and mix thoroughly by vortexing. (A white precipitate may form on addition of ethanol).
  6. Assemble spin column with one of the provided collection tubes and transfer the mixture (including the white precipitate) into the Spin Column and spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
  7. Add 500 μl Buffer W1 (Ensure ethanol (96-100%) has been added) into the Spin Column, spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
  8. Add 700 μl Buffer W2 (Ensure ethanol(96-100%)has been added) into the Spin Column,spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
  9. Spin at 12000 rpm for 2 min and keep the Spin Column at RT for a few minutes to thoroughly dry the column.
  10. Place the Spin Column in a new clean 1.5 ml centrifuge tube, add 50-200 μl pre-warmed Elution Buffer(65°C)directly to the center of the membrane,and incubate at RT for 2-5 min (The volume of eluted buffer should more than 50 μl amd distilled water (pH > 7.0) also can be used to elute gDNA).
  11. Spin at 12000 rpm for 2 min to elute DNA. (The yield can be improved by pipetting the elution back onto the column and repeating step 11.)

B. DNA Extracted From Mouse Tail

  1. Cut 0.4-0.6 cm of fresh or thawed rat tail (0.4-1.2 cm for mouse), and add 180 μl Buffer A.
  2. Continue with step 2 in A.

C. DNA Extracted From Cultured Cells

  1. Centrifuge the appropriate number of cells (no more than 5×106) for 5 min at 300×g. Resuspend the pellet in 200 μl PBS.(PBS provided by users. For frozen cells, resuspend the cells in 200 μl PBS after thaw)
  2. Add 20 μl Proteinase K, mix thoroughly by vortexing.
  3. Continue with step 3 in A.

D. DNA Extracted From Whole Blood

  1. Use 50-200 μl whole blood from mammalian, or 5-10 μl whole blood from amphibian or aves.
  2. Add 20 μl Proteinase K, mix thoroughly by vortexing.
  3. Add PBS up to 220 μl, mix thoroughly by vortexing. (If 200 μl whole blood used, go to next step without adding PBS)
  4. If RNA-free genomic DNA is required, add 4 μl RNaseA (100 mg/ml) and mix the sample by inverting the tube 2~5 times. Incubate the mixture at RT for 2 minutes. (Optional)
  5. Add 200 μl Buffer B, mix thoroughly by vortexing. Incubate at 56℃ with occasional inverting the tube for 10 min.
  6. Continue with step 5 in A.

Troubleshooting

The yield of genomic DNA is low

This may caused by incomplete lysis of cells or the sample is old/overgrown.
  1. Incomplete lysis of cells. Try to reduce the amount of samples,too many cells may result in incomplete lysis of cells and also clogging the spin column.
  2. The sample is old. It may be necessary to use fresh samples. If you need to process the preparation another day, pellet the cells, remove the culture medium and store the pellet at -80°C.
  3. Make sure ethanol (96-100%) has been added into Buffer W1 and Buffer W2.

Downstream application failure with gDNA eluate

Keep the Spin Column at room temperature for a few minutes to dry thoroughly the column before adding elution buffer and the elution buffer should be added directly to the center of the membrane.

Datasheet Download

Genomic DNA Extraction Kit

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