Contents of the Kit
|Lysis buffer ||:100 ml x 1 bottle ||Proteinase K solution ||:1 ml x 2 tubes |
|Precipitation solution I ||:20 ml x 1 bottle ||RNase solution ||0.5 ml x 1 tube |
|Precipitation solution II ||:22 ml x 1 bottle || || |
Storage Condition: 0-5ºC
Shipping Condition: ambient temperature
Required Equipment and Materials
microcentrifuge, vortex mixer, 100 μl and 100-1000 μl adjustable pipettes, ethanol, homogenizer (for tissue), 65oC water bath (for tissue)
Get pureDNA Kit-Cell, Tissue enables isolation and purification of genomic DNA from cell cultures and tissue samples in three easy steps: 1) sample lysis, 2) removal of RNA and protein, and 3) DNA recovery using ethanol precipitation. To isolate genomic DNA from 1 x 108 cells or a 1 g tissue sample, simply increase the volume of each solution (protocols for large samples are available at www.dojindo.com). This kit does not require phenol, chloroform, centrifugal columns, or Filtration tubes. The isolated genomic DNA can be utilized for downstream experiments such as restriction enzyme digestion, ligation, PCR, and other enzymatic reactions.
Protocol for 25 to 30 mg Tissue Sample
1. Transfer 25-30 mg tissue sample into 1.5 ml tube. Add 400 μl Lysis buffer and 10 μl Proteinase K solution.
2. Homogenize the tissue sample using a homogenizer and incubate at 65ºC for 10 minutes.
- or -Incubate at 55ºC for 2-3 hours with occasional vortexing or pipetting (no need for homogenizing).
3. Leave the tube at room temperature for 2 minutes. Add 2 μl RNase solution and vortex for 5 seconds. Leave the tube at room temperature for 2 minutes.
4. Add 80 μl Precipitation solution I and vortex for 5 seconds. White precipitate should appear immediately.
5. Add 80 μl Precipitation solution II and vortex for 5 seconds. More white precipitate should appear. Leave the tube at room temperature for 2 minutes.
6. Centrifuge at 12,000-14,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5 ml tube. To avoid contamination, do not disturb the white precipitate during the supernatant transfer. If white precipitates remain in the transferred supernatant, repeat this step.
7. Add an equal volume of ethanol to the supernatant and mix by inverting the tube several times, then vortex for 5 seconds.
8. Centrifuge at 6,000 rpm for 2 minutes. Carefully remove the supernatant without disturbing the precipitate. A white pellet should be visible at the bottom of the tube. Carefully remove as much of the supernatant as possible.
9. Add 1 ml 70% ethanol and vortex for 5 seconds. Centrifuge at 6,000 rpm for 2 minutes, and discard the supernatant. A white pellet should be visible on the bottom of the tube. Carefully remove the supermatant as much as possible.
10. Dry the DNA pellet using a vacuum desiccator for 10 minutes. Dissolve the pellet in TE buffer and use for downstream experiments. The DNA solution can be stored at 4ºC over one year without degradation.
Table 1 Typical Amount of DNA Recovered from Given Sample TypesThe A280 nm/A260 nm ratio of the recovered DNA is between 1.7 and 1.9.
♦ No or low DNA recovery
a) Completely dissolve the tissue sample in step 2.
b) Make sure there is a DNA pellet at the bottom of the tube before discarding the supernatant in steps 8 and 9.
♦ Difficulty dissolving the tissue sample in step 2
a) Mince or cut the tissue sample into small pieces before transferring it into a tube.
Vortex or pipette every 30 minutes during the incubation. If pipetting,
use a pipette tip with the point cut off, and pipette gently.
♦ Too much precipitate after centrifugation in step 6
a) Completely dissolve the tissue before adding Precipitation solution I.
b) Thoroughly mix the solution by inverting the tube several times after adding precipitation solutions I and II.
c) Increase the centrifugation time if 12,000-14,000 rpm (10,000 g) centrifugation is difficult to achieve.
♦ Low DNA purity
a) Be sure to incubate at room temperature for 2 minutes after adding RNase solution in step 3.