Genomic DNA Extraction Kit (Tissue)
No:DMGTS100 & 300
For research use only
Sample: 30 mg of fresh animal tissue or 25 mg of paraffin-embedded tissue
Format: spin column
Operation time: within 60 minutes
Introduction
The BioDiamond Genomic DNA Extraction Kit (Tissue) was designed specifically for genomic DNA isolation from animal tissue samples. Its unique buffer system ensures total DNA with high yield and good quality from samples while its spin column system purifies or concentrates DNA products previously isolated with buffers. The entire procedure can be completed within 1 hour without phenol / chloroform extraction. Purified DNA is then suitable for use in PCR or other enzymatic reactions.
Kit Contents
NOTE
★ Add ethanol (96–100%) to Buffer W2, shaking before use (see bottle label for volume).
★ Check Buffers before use for salt precipitation. Redissolve any precipitate by warming to 37°C.
★ Buffers W1 contain irritants. Wear gloves when handling these buffers.
Quality Control
In accordance with FairBiotech’s ISO-certified Total Quality Management System, the quality of the BioDiamond Genomic DNA Extraction Kit (Tissue) is tested on a lot-to-lot basis to ensure consistent product quality.
Additional requirements
*RNase A (50 mg/ml) *Proteinase K (10 mg/ml) *mortar and pestle *absolute ethanol *microcentrifuge tubes *micropestle.
Protocol
Step 1 Sample Preparation
Fresh Tissue
- Cut off 30 mg of fresh animal tissue and grind the sample under liquid nitrogen to a fine powder using a mortar and pestle and transfer it to a 1.5 ml microcentrifuge tube or transfer the tissue to a 1.5 ml microcentrifuge tube and use the micropestle to grind the tissue to a pulp.
- Slice small sections (up to 25 mg) from blocks of paraffin-embedded tissue and transfer to a 1.5 ml microcentrifuge tube.
- Add 1 ml of xylene to the tube. Vortex vigorously and incubate at room temperature for approximately 10 minutes. Vortex occasionally during incubation.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Add 1 ml of absolute ethanol to wash the sample pellet and mix by inverting.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Add 1 ml of absolute ethanol to wash the sample pellet again and mix by inverting.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Open the tube and Incubate at 37ºC for 15 minutes to evaporate any ethanol residue.
- Proceed with the Lysis Step.
- Add 300 µl of TL Buffer and 20 µl of Proteinase K( 10mg/ml ) to the tube from Step 1.
- Incubate at 60˚C for 30 minutes and invert the tube every 5 minutes. If the lysate has not become totally clear at the 30 minute mark, use a micropestle to grind the remaining pellet and place the sample back at 60˚C until it is clear. #Pre-heat the Elution Buffer to 60ºC for Step 6 DNA Elution.
RNA Degradation (If RNA-free genomic DNA is required, perform this optional step.)
- Add 5 µl of RNase A (50 mg/ml) to the sample lysate and mix by vortex. Incubate at room temperature for 5 minutes.
- Add 100 µl of TP Buffer to the sample from Step 2 and shake vigorously.
- Centrifuge at 14,000 x g for 3 minute. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
- Add 300 μl of absolute ethanol to the sample lysate and shake vigorously (break up any precipitate by pipetting but be careful to not let any pellet remain inside the pipette tip after you are done).
- Place a TC Column in a 2 ml Collection Tube.
- Transfer the mixture completely from the previous step to the TC Column.
- Centrifuge at 14,000 x g for 30 seconds.
- Discard the flow-through and place the TC Column back in the 2 ml Collection Tube.
- Add 400 µl of W1 Buffer into the TC Column. Centrifuge at 14,000 x g for 30 seconds.
- Discard the flow-through and place the TC Column back into the same Collection tube.
- Add 600 µl of W2 Buffer (Ethanol added) into the TC Column. Centrifuge at 14,000 x g for 30 seconds.
- Discard the flow-through and place the TC Column back into the same Collection tube.
- Centrifuge at 14,000 x g again for 2 minutes to remove residual W2 Buffer.
- Transfer the dried TC Column to a new 1.5 ml microcentrifuge tube.
- Add 50-200 µl of Pre-Heated EL Buffer or TE into the center of the column matrix.
- Let stand at 60ºC for 3 minutes.
- Centrifuge for 2 minutes at 14,000 x g to elute the purified DNA.
Troubleshooting
dmgts100___300.pdf | |
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