Method for extracting total genomic DNA from acidulated heavy metal tailings
A heavy metal and genome technology, applied in recombinant DNA technology, DNA preparation and other directions, can solve problems such as ineffective extraction, and achieve the effects of obvious PCR specific products, clear DNA bands, and simple operation.
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Embodiment 1
[0053] Embodiment 1, extracting the total DNA of acidified heavy metal tailings genome, including steps:
[0054] (1) Take about 0.5g of acidified heavy metal tailings, put it in a 1.5mL centrifuge tube, add 1mL TE1 buffer, vortex for 5min, centrifuge at 6000r / min for 5min, discard the supernatant, then add 1mlTE1 buffer, vortex Shake for 5 minutes, centrifuge at 6000r / min for 5 minutes, and discard the supernatant.
[0055] (2) Add 600 μL of TE2 extraction buffer and 10 μL of lysozyme (100 mg / mL) into a constant temperature water bath at 37°C for 1 hour, and mix by inverting every 10 minutes.
[0056] (3) Cool in liquid nitrogen for 5 minutes, quickly put it in a boiling water bath for 5 minutes, and repeat this 3 times.
[0057] (4) Add 120 μL of 20% SDS and 10 μL of 100 mg / mL proteinase K, put it into a constant temperature water bath at 65°C for 1 hour, and mix it by inversion every 10 minutes during this period. Lowered to room temperature, centrifuged at 8000r / min for ...
Embodiment 2
[0065] Embodiment two, determine the concentration and the purity of the total genomic DNA in the sample:
[0066] Utilize the ultraviolet spectrophotometer to detect the concentration and the purity of DNA, measure the absorbance value of the DNA solution that extracts respectively at 260nm, 280nm, the absorbance value that the inventive method extracts DNA is as shown in table 1, the DNA that traditional soil DNA extraction method obtains The absorbance value is shown in Table 2, and the concentration and purity of the DNA sample are calculated.
[0067] The purity and concentration of the acidified heavy metal tailings genome total DNA extracted by the method of the present invention in table 1
[0068]
[0069] Table 2 The purity and concentration of the acidified heavy metal tailings genome total DNA extracted by traditional soil DNA extraction methods in the prior art
[0070]
[0071] Conclusion: Compared with the traditional soil DNA extraction method in the pri...
Embodiment 3
[0072] Example 3, PCR product detection of 16S rRNA of acidified heavy metal tailings genome total DNA:
[0073] Use bacterial universal primers 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3' and 1492R: 5'-GGTTAC CTT GTT ACG ACT T-3'; the total volume of the PCR reaction system is 50 μL, 25 μL Mix (10×Buffer, dNTP , Mg 2+ , TaqDNA polymerase), 2 μL DNA template, 2 μL each primer, 19 μL sterile double distilled water. PCR reaction system: Denaturation at 94°C for 60s, annealing at 50°C for 30s, extension at 72°C for 60s, 35 cycles. Take 5 μL of the product and detect it by electrophoresis in 0.8% agarose gel.
[0074] Result: if image 3 As shown, the samples were effectively amplified, the bands of the amplified products were clear, the fragments were about 1500bp, and the specific fragments were of the same length, indicating that the total genomic DNA in the acidified heavy metal tailings extracted by this method can meet the requirements of subsequent PCR experiments.
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