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Universal DNA Purification Kit 

For the extraction of DNA fragments from agarose gels, PCR and enzymatic reactions.
Cat. No. Size Price Quantity  
TB50002A 50 preps USD 89
TB50002B 200 preps USD 280
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

Contents 50 preps
Buffer PD 50 ml
Buffer PW 15 ml
Buffer EB 5 ml
Spin Columns 50
Collection Tubes 50

Storage and Stability

The Universal DNA Purification Kit can be stored for up to 12 months at room temperature (15−25℃) or at 2−8℃ for storage periods longer than 12 months. Check buffers for precipitate before use and redissolve at 37℃ if necessary.

Description

The Universal DNA Purification Kit is designed for efficient and convenient purification of DNA fragments from agarose gels, PCR and enzymatic reactions. The kit utilizes a silica-based membrane system in the form of a spin column, which can bind up to 10 µg of DNA selectively. Special buffers are optimized for efficient recovery of DNA and removal of proteins, salts, and organic molecules. The kit can be used to purify DNA fragments from 100 bp−10 kb in size, and recovery rates are 60%-80% in this size rage. DNA purified with Universal DNA Purification Kit is suitable for many subsequent applications, such as restriction, ligation, PCR, sequencing, labeling, in vitro transcription and microinjection.

Important notes before starting

  1. Add 60 ml ethanol (96-100%) to buffer PW before use, and label the bottle accordingly.
  2. TAE or TBE buffer should be fresh.
  3. When you cut the DNA fragment, you should avoid damaging the DNA through UV light exposure and cut as close to the DNA as possible to minimize the gel volume.
  4. The yellow color of Buffer PD indicates a pH≤7.0.
  5. All centrifugation steps are carried out at room temperature(15−25℃).
  6. The Buffer PD contains strong denaturant. When working with the Buffer PD, always wear suitable protective clothing such as safety glasses, laboratory coat and gloves. Be careful to avoid contact with eyes and skin. In the case of such contact, wash immediately with water.

Procedure

DNA extraction from agarose gels

  1. Excise the DNA fragment from an agarose gel. Remove extra agarose.
  2. Weigh the gel slice in a clean tube. Add 3 times volumes of Buffer PD to 1 volume of gel (100 mg-100 µl). Incubate at 65°C for 10 min. Vortex the sample briefly every 2−3 mins until the gel slice is completely dissolved.
    • For example, add 300 µl Buffer PD to each 100 mg of gel. For gels containing >2% agarose, add 6 volumes of Buffer PD.
  3. After the gel slice has dissolved completely, check that the color of the mixture is yellow, similar to Buffer PD without gel slice.
    • If the color of the mixture is pink or violet, add 10−20 µl 3 M sodium acetate pH 5.0, and mix. The color of the mixture will turn yellow.
    • When the DNA fragment <500 bp, add same volume of isopropanol with the gel to increase the yield of the DNA.
  4. Place a spin column into a provided collection tube (2 ml) and load up to 750 µl sample. Centrifuge for 1 min at 12,000×g. Discard flow-through and place the column back to the collection tube.
    • For sample volumes of more than 750 µl, simply load and centrifuge again.
  5. Add 700 µl buffer PW to the spin column. Centrifuge at 12,000×g for 1 min. Discard flow-through and place the column back to the collection tube.
  6. Repeat step 5.
  7. Centrifuge the spin column for an additional 1 min at 12,000×g to remove Buffer PW.
    • Make sure that the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
  8. Place the spin column into a clean 1.5 ml microcentrifuge tube (not provided). Open the lid, and stand in the air for 1 min to evaporate the liquid. To elute DNA, add 50 µl of Buffer EB or deionized water (pH 7.0−8.5) to the center of the membrane and incubate at room temperature (15−25℃) for 1 min. Centrifuge at 12,000×g for 1 min.
    • Pre-warming the Buffer EB at 60°C will generally improve elution efficiency.
    • For increased DNA concentration, add 30 µl of Buffer EB to the center of the membrane.

DNA extraction from PCR and other enzymatic reactions

  1. Mix the sample with 5 times volumes of Buffer PD.
    • For example, mix 100 µl PCR reaction and 500 µl Buffer EB.
    • For very small sample volumes <100 µl adjust the volume of the reaction mixture with water.
    • When the DNA fragment <500 bp, add same volume of isopropanol with the gel to increase the yield of the DNA.
  2. Place a spin column into a provided collection tube (2 ml) and load up to 750 µl sample. Centrifuge at 12,000×g for 1 min. Discard flow-through and place the column back to the collection tube.
    • For sample volumes of more than 750 µl, simply load and centrifuge again.
  3. Continue with Step 5 of the protocol “DNA extraction from agarose gels”.

Troubleshooting

Low recovery

  1. Incompletely dissolved gel slice

    Increase time or add more Buffer PD. Vortex the tube briefly every 2−3 minutes during heating to enhance solubilization.

  2. Reagent not prepared properly

    Add indicated volume of 96%−100% ethanol to Buffer PW and mix well before use.

  3. Elution Buffer incorrectly dispensed.

    Add the Buffer EB to the center of the spin column membrane to ensure that the buffer completely covers the membrane.

  4. Sample mixture turns pink or violet

    If sample mixture turns pink or violet, add 10-20 µl 3 M sodium acetate pH 5.0 to the mixture to turn the color back to yellow.

DNA purified does not perform well

  1. High salt concentration

    Modify the wash step by incubating the spin column for 5 mins at room temperature after adding 750 µl of Buffer PW, then centrifuge. Use the deionized water (pH 7.0−8.5) for elution.

  2. Residual ethanol

    Before elution, centrifuge additional for 1 min at 12,000×g and incubate the spin column at room temperature for 5 mins to remove ethanolic Buffer PW completely.

Datasheet Download

Universal DNA Purification Kit

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