订货Q Q： 247937283、1098867025
AipBest Soil Genome DNA ExtractionKit（Bead Beating）
The AipBest SoilGenome DNA Extraction Kit quickly and efficiently isolates PCR-ready genomicDNAdirectly from soil samples in less than 40 minutes. Designed for use withBeads-Beating device such as the FastPrep® Instruments from MP Biomedicals，soil organisms population are easily lysed within 40 seconds. Samplesare placed into 2.0 ml tubes containing 3 kinds of beads, a mixture of ceramicand silica particles designed to efficiently lyse all soil organisms includinghistorically difficult sources such as eubacterial spores and endospores, grampositive bacteria, yeast, algae, nematodes and fungi.
The kit uses a novel and proprietary method to remove high humic acidcontent including difficult soil types such as compost, sediment, andmanure.The isolated DNA has a high level of purity allowing for more successfulPCR amplification of organisms from the sample. PCR analysis has been performedto detect a variety of organisms including bacteria (e.g. Bacillus subtilis,Bacillus anthracis), fungi (e.g. yeasts, molds), algae and Actinomycetes (e.g. Streptomyces).
◆Important consideration before use
The fill volume in the bead tube after the addition of the SodiumPhosphate and MT Buffers to the sample should allow sufficient air space in thesample tube for efficient FastPrep® Instrument processing. MP Biomedicalsrecommends using 500 mg of starting material as long as there is between 250 -500 μl of empty spacein the tube. Sample loss or tube failure may result from overfilling the beadtube. The bead tube caps must be secure, but not over-tightened, to preventsample leakage. If the sample is too large for processing in a single tube,divide the sample and process using multiple tubes. The kits have beenrigorously tested in the FastPrep® Instrument. A single 40 second run at aspeed setting of 6.0 in the FastPrep® Instrument is sufficient to lyse almostall samples. If the user experimentally determines that additional processingtime is required, the sample should be incubated on ice in the bead tube for atleast 2 minutes between successive FastPrep® Instrument homogenizations toprevent overheating the sample and tube.
If you use other bead beater device, please follow instruction manualfrom manufaturer to set appropriate parameter for good performance.
ð Before thefirst use, add the indicated amount of ethanol into PQ solution bottle, Buffer WBbottle, mix well, and mark the bottle with a check.
1. Add up to 500 mg ofsoil sample to a Bead Tube.
2. Add 980 μl Sodium Phosphate Buffer to sample in BeadTube. Gentle votex to mix. Add 120 μl MT Buffer.
Note: Check MT Buffer. If MT Buffer is precipitated, heat solution to 60°Cuntil dissolved before use.3. Homogenize in theFastPrep® Instrument for 40 seconds at a speed setting of 6.0.
4. Centrifuge at 12,000x g for 5minutes to pellet debris.
5. Transfer supernatantto a clean 2.0 ml centrifuge tube. Add 250μl PPS Solution and mix by shaking the tube byhand 10 times. Incubate at 4°C for 5 minutes.
6. Centrifuge tubes at10,000 x g for 3 minute at room temperature. Avoiding pellet, transfer up to,but no more than, 900 μl ofsupernatant to a clean 2 ml centrifuge tube.
7. Add 300 μl of IRS Solution(1/3 volume) and vortexbriefly. Incubate at 4°C for 5 minutes.
8. Centrifuge tubes at10,000 x g for 1 minute at room temperature. Avoiding pellet, transfer the supernatant into a clean 5 ml centrifugetube.
9. Add 1.5 volumes of PQSolution to the cleared supernatant and mix by pipetting.
Example: To 1100 μl lysate add 1650 μl PQ Solution. Reduce the amount of PQSolution accordingly if less supernatant is recovered. A precipitate may formafter the addition of ethanol but this will not affect the procedure.
Note: Ensure ethanol has been added to PQ Solution.
Note: It is important to pipet PQ Solution directly onto the cleared supernatantand to mix immediately.
10. Load approximately700 μl mixture onto Spin Filter(sitting in collection tube) and centrifuge at 10,000 x gfor 1 minute at room temperature. Discard flow through. Load another 700 μland repeat untill all remaining mixtureis loaded on Spin Filter.
Note: A total of 4-5 loads for each sample processed may be required.
11. Add 600 μl of BufferWB to Spin Filter and centrifuge at 10,000 x g for 30 seconds at roomtemperature. Discard flow through. RepeatStep 11 with another 600 µl Buffer WB.
Note: Ensure ethanol is added to Buffer WB.
12. Centrifuge SpinFilter at 13,000 x g for 2 minute at room temperature to dry the Spin Filter..
13. Carefully place SpinFilter in clean 1.5 ml centrifuge tube. Avoid splashing any Buffer WB onto SpinFilter. Add 100 µl of Elution Buffer (Optional:pre-warm the water to 70–90°C will increase the DNA yield) to the center of the column membrane.Incubate at room temperature for 3-5 min, and centrifuge at 13,000 x g for 1min to elute the DNA.
Note: Use smaller volume(minimum 30µl)of Elution Buffer will obtain higher concentration.
Optional: Put eluate back to the SpinColumn to repeat elution once. This increases concentration of DNA about10-15%.