morgan@go2biotech.comGDP613-DE TGuide Smart Envir-DNA Kit
For purification DNA from various environmental samples such as soil, stool, fermentation products,water, pond sludge, marine sediment, and intestinal contents, etc.
TECHNICAL MANUAL
Cat. no. GDP613-DE
Note: To use the TGuide Smart Envir-DNA Kit, you must have the TGuide Smart
Envir-DNA program ( No. DP613) installed on the TGuide S16/S32/S32 Pro/S96
Dex Nucleic Acid Extractor.
Table Contents
Kit Contents
Envir-DNA reagents composition
Storage
Introduction
Features
Notes
Protocol
（Prefilled single sample cartridge）
（Suggested dosage for different samples）
(Sample pre-treatment )
(TGuide S16/S32/S32 Pro/S96 Dex Automated Extraction Procedure )
Appendix
TGuide Smart Envir-DNA Kit
Cat. no. GDP613-DE
Introduction
This kit uses a unique lysis solution combined with a specially optimized and highly
efficient HA removal buffer to extract genomic DNA of Gram-negative bacteria, Grampositive bacteria, fungi and other microorganisms from a variety of environmental
samples, such as soil, feces, fermentation products, water, pond sludge, marine
sediments, and intestinal contents. The grinding beads and homogenizer can
effectively crush the microorganisms in the environmental samples to ensure the
high efficiency and integrity of the extracted genomic DNA.
Genomic DNA purified by this kit has few impurities and good integrity, and can be
directly used in downstream experiments of molecular biology such as PCR, library
prep and NGS.
Features
Quick and easy: Quickly remove humic acid in 1 min.
High purity:Combined with magnetic bead purification, the extracted DNA has high
purity and can be directly used in downstream experiments.
Wide application: Can be applied to microbial genomic DNA extraction for many
types of samples.
Good compatibility: It can be applied to a wide range of TGuide S16/S32/S32 Pro
automated nucleic acid extractor and can be extended for use with the TGuide S96
Dex Automated Nucleic Acid Extractor.
Notes ：Please be sure to read this precaution before using the kit.
- The newly collected samples will get higher yield, and different samples should
consult the corresponding optimal preservation conditions before sampling. - Excess DNA may inhibit downstream PCR reactions, and it is recommended to
dilute the DNA template for use if inhibition appeared. - HA Removal Buffer CN needs to be pre-cooled at 2-8°C before use in order to
achieve its optimal humic acid removal effect. - Buffer TB does not contain EDTA. In order to minimize the degradation of the
extracted nucleic acids for long time storage, customer can replace the Buffer
TB with the EDTA-containing Buffer TE if necessary, but pay attention to the
fact that the EDTA will affect the downstream experiments, such as PCR, library
construction, and so on. - Due to the complexity of environmental samples, which usually have more
impurities, the concentration of extracted nucleic acids is low, and degradation of
nucleic acids may occur in long time storage, etc. It is recommended to carry out
downstream experiments as soon as possible after extraction. - This kit is equipped with two elution buffers. Choose corresponding one for the
blank control.
Protocol
- Prefilled single sample cartridge
1) Take out a prefilled single sample cartridge and invert it to re-suspend the
magnetic beads; Gently shake to concentrate the reagent and magnetic beads to
the bottom of the cartridge. Before use, remove the sealing film carefully to avoid
liquid spatter or spills.
2) Add the Buffer TB/Buffer TE of appropriate volume (60~100 μl) into the 5th well of
the single sample reagent cartridge. - Suggested dosage for different samples
1) For common soil or fecal samples: Take 250 mg of soil samples (loam, clay,
subsoil, garden soil, farmland soil, etc.) and 100 mg of fecal samples (e.g.,
cattle and sheep feces, poultry feces, poultry intestinal contents, fish and shrimp
intestinal contents, etc.) in Envir-DNA Grinding Beads Tubes; for fecal samples
preserved in alcoholic preservation solution, take an appropriate amount of the
sample (not more than 100 mg of the dry mass) by high-speed centrifugation
to remove the preservation solution. For fecal samples containing alcohol
preservative, take an appropriate amount of sample (dry matter mass not more
than 100 mg) and centrifuge it at high speed to remove the preservative, then
transfer it to the Envir-DNA Grinding Beads Tube.
Note: If the soil or fecal sample is high in impurities and rich in microorganisms,
reduce the sample input appropriately. Otherwise, it may result in varying degrees
of color residue in the eluate.
2) Samples with high water content: e.g. fermentation broth, cellar yellow water,
washed honey or nectar, milk, etc., take an appropriate volume of sample,
centrifuge at 12,000 rpm (~13,400×g) for 1-2 min, remove the supernatant, and
collect the precipitate amount of about 10-150 mg, or when there is no significant
precipitation after centrifugation, 50-100μl of liquid can be retained in the aspirate
when discarding the supernatant and then add the 600μl Buffer SNA, resuspend
and mix well, then transfer to Envir-DNA Grinding Beads Tube and proceed to the
next step.
3) Aqueous or Air Filter Membrane: Cut up the filter membrane, 47 mm diameter filter
membrane is cut up and put into 2 ml Envir-DNA Grinding Beads Tube, 147 mm
diameter filter membrane is cut up and can be dispensed into 2-3 tubes of 2 ml
Envir-DNA Grinding Beads Tubes or 1 tube of 5ml Grinding Beads Tube.
Note: Only 48×2 ml Envir-DNA Grinding Beads Tubes are included in the kit, if
more 2 ml Envir-DNA Grinding Beads Tubes or 5 ml Grinding Tubes are needed,
the customer needs to bring their own.
4) Highly absorbent samples: e.g., powdered dacquoise, etc. Weigh 100 mg of
sample in Envir-DNA Grinding Beads Tubes.
5) Other special samples:
a) Honey: take 10-15 g of honey, add 3-4 times the volume of pure water (provided
by the customer), mix well, centrifuge at 8,000 rpm (~6,000×g ) for 10 min,
remove the supernatant, add 1ml of pure water (provided by the customer)
to resuspend the precipitate, centrifuge at 8,000 rpm (~6,000×g ) for 10 min,
remove the supernatant, and then add 600 μl of Buffer SNA, resuspend the
precipitate and transfer to Envir-DNA Grinding Beads Tube for the next step.
b) Fruiting body of fungal, tissue samples, intestinal contents containing intestinal
tissues: take 10-100 mg samples and cut them into Envir-DNA Grinding Beads
Tube. - Sample pre-treatment
1) Weigh the sample into the Envir-DNA Grinding Beads Tube according to the
procedure “2. Suggestions for different sample amounts”.
2) Add 600-700 µl of Buffer SNA and 10 µl of RNase A (10 mg/ml), vortex and mix to
thoroughly break up and resuspend solid samples in solution, homogenize with
TGrinder H24 and homogenize with a program of 20 sec of oscillation at 6 m/s
with 20 sec intervals for a total of 6 cycles.
Note:
a) For samples that have been recommended to add Buffer SNA in “2.
Recommendations for different samples”, it is not necessary to add Buffer
SNA in this step, only 10 µl of RNase A should be added.
b) For highly absorbent samples, the amount of Buffer SNA can be increased
so that the volume of supernatant obtained by centrifugation after
homogenization is 400-500 µl.
c) For larger sample quantities, a proportional mixture of Buffer SNA and RNase
A can be prepared freshly.
d) Drier stools, denser or centrifuged samples need to be vortex and broken
up as much as possible after the buffer is added, otherwise the subsequent
homogenization will be affected;
e) If a tissue homogenizer is not available, a vortexer with high speed for 10 min
or an oscillator with 50 Hz for 10 min can be used instead of homogenization.
f) If the integrity of the genomic bands is required to be high, the oscillation
speed of the homogenization procedure can be reduced to 4 m/s or the
number of cycles can be reduced to 2 cycles (the length of the vortexer and
the oscillator can be shortened to 5 min or the oscillator speed can be reduced
to 25 Hz), in which case the integrity of the genomic bands will be good, but
the extraction rate will be slightly reduced.
3) (Optional step) After homogenization, oscillate for 10 min at 70°C 1,200 rpm on a
thermostatic metal bath.
Note: Heating is not recommended for samples with dark color and high content
of humic acid and other impurities to prevent the release of too many impurities
and affect the purity of nucleic acid extraction.
4) Centrifuge at 12,000 rpm (~13,400×g) for 1 min and transfer the supernatant
(~400-500 µl) to a 1.5ml centrifuge tube.
5) Add 200 μl of pre-cooled HA Removal Buffer CN at 2-8°C, vortex for 3-5 sec, and
centrifuge at 12,000 rpm (~13,400×g) for 1 min.
Note:
a) When the content of microorganisms and inhibitors in the sample is low, the
amount of HA Removal Buffer CN can be reduced to 100 µl, and HA Removal
Buffer CN cannot be added to the clarified liquid samples to improve the
extraction rate.
b) When the content of impurities and inhibitors is high, the amount of HA
Removal Buffer CN can be increased to 300 µl, which can improve the purity of
nucleic acids. - TGuide S16/S32/S32 Pro/S96 Dex Automated Extraction Procedure
1) Transfer 500 µl of the processed supernatant from step “3. Sample pre-treatment”
to well 1.
2) Place the reagent tank bracket on the plate base in the TGuide S16/S32/S32 Pro/
S96 Dex Automated Nucleic Acid Extractor, insert the tip comb into the slot to
ensure they are well connected and firmed.
3) Select the corresponding program and press the Start button to begin running the
extraction program.
Enter the program of the TGuide S16/S32/S32 Pro/S96 Dex, select the “DP613”
experimental program file and click the Run button to start the experiment.
4) At the end of the automated extraction process, take the DNA out from the 5th well
of the cartridge and store it under appropriate conditions. Single sample reagent
cartridge and tip comb are for single use only. It is recommended to store the
eluted product at -30~-15℃ or -90~-65℃
Appendix
Table 1: TGuide S16 Automatic Nucleic Acid Extraction and Purification Instrument
Environmental Microbial Genomic DNA Extraction Procedure
Table 2: DNA extraction procedure for environmental microbial genome of TGuide
S32/S32 Pro fully automated nucleic acid extraction and purification instrument
The automated nucleic acid extraction procedure is shown in the table below:
Table 3: TGuide S96 Dex Fully Automated Nucleic Acid Extraction and Purification
Instrument Environmental Microbial Genomic DNA Extraction Procedure
GDP613-DE TGuide Smart Envir-DNA Kit
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