GDP302-TIANamp Bacteria DNA Kit

TIANamp Bacteria DNA Kit—— For isolation of genomic DNA from bacteria

TIANamp Bacteria DNA Kit
(Spin Column)
Cat.no. GDP302

Introduction
TIANamp Bacteria DNA Kit is based on silica membrane technology and
provides special buffer system for many kinds sample of gDNA extraction.
The spin column is made of new type of silica membrane which can bind
DNA optimally on given salt and pH conditions. Simple centrifugation
processing completely removes contaminants and enzyme inhibitors such
as proteins and divalent cations. Purified DNA is eluted in low-salt buffer or
water, and is ready for use in downstream applications.
DNA purified by TIANamp Bacteria DNA Kit is highly suited for restriction
enzyme digestion, PCR analysis, Southern blotting, and cDNA library.

Features

1. Simple and fast:Pure genomic DNA of Gram-negative bacteria can be
   obtained within 1 hour.
2. Excellent quality: The purified DNA can be directly used in downstream
   molecular experiments such as PCR, restriction endonuclease digestion,
   Southern blotting, etc.

Important Notes Please carefully read before using this kit.

1. Repeated freezing and thawing of stored samples should be avoided,
   since this leads to DNA size reduction.
2. If precipitates formed in Buffer GA or Buffer GB, warm the buffer to
   37°C until the precipitates have fully dissolved.
3. All centrifugation steps should be carried out in a conventional tabletop centrifuge at room temperature (15-30°C).

Reagents need to be prepared by Customer
96-100% ethanol, RNase A (100 mg/ml) (optional); Lysozyme (50 mg/ml)
(TIANGEN) (optional)

Protocol
Ensure that Buffer GD and Buffer PW have been prepared with appropriate
volume of ethanol (96%-100%) as indicated on the bottle and shake thoroughly.

1. Pipet 1-5 ml bacterial culture suspension in a centrifuge tube, centrifuging for 1
   min at 10,000 rpm (~11,500 × g), discard supernatant as possible.
2. Add 200 μl Buffer GA. Mix thoroughly by vortex.
   Note: For Gram-positive bacteria, Step 2 could be replaced by lysozyme
   treatment: add 110 μl enzymatic lysis buffer (20 mM Tris·Cl, pH 8.0; 2 mM
   sodium EDTA; 1.2% Triton® X-100), and 70 μl lysozyme (50 mg/ml prepared
   by users). Incubate for at least 30 min at 37°C.
   If RNA-Free genomic DNA is required, add 4 μl RNase A (100 mg/ml, should
   be prepared by user), mix by vortex for 15 sec, and incubate for 5 min at
   room temperature (15-30°C).
3. Add 20 μl Proteinase K. Mix thoroughly by vortex.
4. Add 220 μl Buffer GB to the sample, vortex for 15 sec, and incubate at 70°C for
   10 min to yield a homogeneous solution. Briefly centrifuge the 1.5 ml centrifuge
   tube to remove drops from the inside of the lid.
   Note: White precipitates may form when Buffer GB is added. They will not
   interfere with the procedure and will dissolve during the heat incubation
   at 70°C. If precipitates do not dissolve during heat incubation, it indicates
   that the cell is not completely lysed and may result in low yield of DNA and
   impurity in DNA.
5. Add 220 μl ethanol to the sample, and mix thoroughly by vortex for 15 sec. A
   white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml
   centrifuge tube to remove drops from the inside of the lid.
6. Pipet the mixture from step 5 into the Spin Column CB3 (in a 2 ml collection
   tube) and centrifuge at 12,000 rpm (~13,400 × g) for 30 sec. Discard flowthrough and place the spin column into the collection tube.
7. Add 500 μl Buffer GD (Ensure ethanol has been added) to Spin Column CB3,
   and centrifuge at 12,000 rpm (~13,400 × g) for 30 sec, then discard the flowthrough and place the spin column into the collection tube.
8. Add 600 μl Buffer PW (Ensure ethanol has been added) to Spin Column CB3,
   and centrifuge at 12,000 rpm (~13,400 × g) for 30 sec. Discard the flowthrough and place the spin column into the collection tube.
9. Repeat Step 8.
10. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to dry the membrane
    completely.
    Note: The residual ethanol of buffer PW may affect downstream
    application.
11. Place the Spin Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet
    50-200 μl Buffer TE or distilled water directly to the center of the membrane.
    Incubate at room temperature for 2-5 min, and then centrifuge for 2 min at
    12,000 rpm (~13,400 × g).
    Note: If the volume of eluted buffer is less than 50 μl, it may affect
    recovery efficiency. The pH value of eluted buffer will have some influence
    in eluting. We suggest choosing buffer TE or distilled water (pH 7.0-8.5) to
    elute gDNA. For long-term storage of DNA, eluting in Buffer TE and storing
    at -30~-15°C is recommended, since DNA stored in water is subject to acid
    hydrolysis.
    To enhance the gDNA yield, eluate can be reloaded to CB3 once, incubate
    at room temperature 2min and centrifuge at 12,000 rpm (~13,400 × g) for 2
    min to get the final eluate.

GDP302-TIANamp Bacteria DNA Kit

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