CN110029104A - A kind of extracting method of the bacillus licheniformis RNA suitable for fermentation process - Google Patents
A kind of extracting method of the bacillus licheniformis RNA suitable for fermentation process Download PDFInfo
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Abstract
The extracting method of the invention discloses a kind of suitable for fermentation process bacillus licheniformis RNA, this method is in the case where having determined different fermentations period microorganism collection ratio, combine common RNA kit (the raw work RNA extracts kit in Shanghai, BioFlux RNA extracts kit, TaKaRa RNA extracts kit, the Hangzhou treasured match biology RNA extracts kit etc.) extraction method and the portable RNA method of tradition of the common microbiologicals such as Escherichia coli, bacillus licheniformis RNA can be significantly improved and extract quality, and bacterium solution liquid needed for RNA extraction is few (50-300 μ L)；The RNA of extraction has many advantages, such as that purity is high, moderate concentration, stability are high, can sufficiently meet the downstream applications such as RT-PCR, RT-qPCR.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to extraction bacillus licheniformis RNA in a kind of fermentation process
Method.
Background technique
Ribonucleic acid (Ribonucleic Acid, RNA) is that one kind is present in biological cell and fractionated viral, class disease
The carrier of hereditary information in poison.It, according to base pair complementarity principle, is transcribed and is formed using a chain of DNA as template
One it is single-stranded, major function is to realize expression of the hereditary information on protein, is hereditary information to Phenotypic
In bridge.Quantitative analysis is carried out by the RNA to microorganism under different conditions, the phenotype tune of organism can be fully understood
Control, knows the expression of gene, and RNA up-to-standard in this kind of research is to ensure that the successful premise of research experiment.
Bacillus licheniformis (Bacillus licheniformis) belongs to gram-positive bacteria, is important industrial microorganism
Bacterial strain, the research in terms of transcription are often limited to the quality of RNA.At present when extracting bacillus licheniformis RNA, biography is used
System method or general reagent cassette method can not obtain good result.It would ordinarily be encountered following situation: first is that the portable side of tradition
The biomass that method needs to collect is bigger (being greater than 10mL), and time-consuming, this often growth to thallus in fermentation process
Cause strong influence with metabolism, cause the accuracy of the experiments such as subsequent RT-PCR, RT-qPCR, transcriptome analysis it is low, reappear
Property is poor.Second is that the bacillus licheniformis intracellular protein Content of different growth conditions is different, also difference is very for the RNA mass of extraction
Greatly, because the quality of RNA can all be reduced by collecting excessive or very few thallus.Third is that during the lichen bacillus ferments, it should
Mycetocyte exocrine protein is very capable, and protein matter is usually attached to phage surface, and the portable method of tradition, which will lead to, to be extracted
Engineered protein residual is excessive, and the deproteinized solution in kit often plays good effect.Fourth is that bacillus licheniformis is thin
Cell wall is thicker, and the chemical reagent that kit provides cracks difficult, currently a popular Tissue Cell-Destroyer DS1000
It needs broken a period of time to stop a period of time in broken wall, takes a long time, be easy the mortar grinder for making RNA degrade, and traditional
Broken wall can be completed in method grinding 30s.Fifth is that bacillus licheniformis will form one layer " mycoderm " or be wadded a quilt with cotton during liquid state fermentation
Shape precipitating, causes the thallus homogeneity collected poor.Generally speaking, there is no for bacillus licheniformis, this is special at present
Gram-positive bacteria illustrates extracting method.
Thallus reaches stationary phase during the fermentation, and biomass increases as the time increases, so according in fermentation process
The OD600 of bacterium solution can determine microorganism collection ratio.Earlier fermentation bacterial metabolism rate is lower, and production protein content is relatively low, therefore
The cell quantity for needing to collect is also more；The later period bacterial metabolism that ferments is active, and protein secretion is high, it is therefore desirable to which appropriate reduce is received
The cell number of collection avoids the influence of foreign protein in RNA extraction process.
Based on background above, on the basis of determining microorganism collection ratio, in conjunction with the portable method of tradition and isolation kit method
Above-mentioned puzzlement can be overcome, obtain up-to-standard RNA, and will not influence in fermentation process the growth of bacillus licheniformis and
Metabolism, it is ensured that the success of subsequent experimental.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of lichens suitable for fermentation process
The extracting method of bacillus RNA.The present invention is suitable for kinds of experiments room and often uses antibacterial agents box, can significantly improve lichens bud
Spore bacillus RNA extracts quality, fully meets the downstream applications such as RT-PCR, RT-qPCR.
Technical scheme is as follows:
A kind of extracting method of the bacillus licheniformis RNA suitable for fermentation process, the extracting method include following step
It is rapid:
(1) according to biomass, the fermentation liquid during the lichen bacillus ferments is collected；
(2) fermentation liquid obtained to step (1) is washed, is crushed, enzymolysis processing；
(3) using kit to by step (2) treated that sample cracked, clean, wash, elute.
The specific steps of the method are as follows:
(1) fermentation liquid is shaken up in fermentation process, 500 μ L of real time sample is placed in 1.5mL Ep pipe, takes 60 μ L bacterium solutions dilute
Fermentation liquid OD600 is measured after releasing 50 times, remaining sample is immediately placed in -80 DEG C of refrigerators and saves；
(2) to which after fermentation, the bacterium solution ice bath 0.5-1h saved in -80 DEG C of refrigerators thaws in advance；
(3) according to the OD600 measured, the thalline quantity in samples taken is controlled in suitable range: (0.2-0.5)
×109Cfu takes bacterium solution (800-2000)/OD600 μ L after thawing in 1.5mL Ep pipe, respectively in room temperature 12000rpm
It is centrifuged 30s；
(4) supernatant is outwelled, 600-800 μ L ddH is added2O, in thallus 30s is resuspended on vortex mixer, in room temperature
12000rpm is centrifuged 30s；
(5) supernatant is outwelled, 1mL ddH is added2O, in thallus 30s is resuspended on vortex mixer, by the bacterium solution after dilution
Be transferred in the mortar that Liquid nitrogen precooler is crossed, add microglass bead and liquid nitrogen, grind into powder, immediately after with key by it is small
The heart is transferred in 1.5mL Ep pipe, and the sample being disposed can place -80 DEG C of preservations.
(6) lysozyme of the 1-5g/L of 100 μ L Tris-HCl buffers is added in step (5) treated sample
Liquid, in thallus 30s is resuspended on vortex mixer, then enzyme digestion reaction 5-10min at room temperature；
(7) according to kit specification, lysate, protein liquid removal, rinsing liquid are sequentially added, wherein deproteinized step needs
It is repeated 2 times, 1min need to be got rid of in sky under 12000rpm after rinsing, then room temperature dries 5min, is eventually adding the RNase- of 30-50 μ L
Free water elution collects RNA；
(8) RNA of 10 μ L is taken to be used to carry out Concentration Testing, nucleic acid electrophoresis, remaining RNA is placed on -80 with liquid nitrogen frozen
It is saved in DEG C refrigerator；
(9) when carrying out a RT-PCR or step RT-qPCR, take the RNA of 0.5 μ L as template, addition reverse transcription enzyme system or
QPCR enzyme system synthesizes cDNA with this and is reacted.
Preservation time of the fermentation liquid described in step (1) in -80 DEG C of refrigerators must not exceed 5 days.
The relational expression of thalline quantity and OD600 described in step (3): according to 1OD600 ≈ 0.25 × 109Cfu/mL meter
It calculates, when fermentation liquid OD600 is less than 20 in fermentation process, collected bacterium solution volume is 2000/OD600 μ L；Work as fermentation
When fermentation liquid OD600 is greater than 30 in the process, collected bacterium solution volume is 800/OD600 μ L.
Microglass bead diameter described in step (5) be 425-600 μm, mortar diameter be 60mm, 80mm, 90mm, 100mm or
130mm。
The bacteriolyze enzyme solution that concentration described in step (5) is 5g/L reacts 5min；The bacteriolyze enzyme solution of 4g/L reacts 6min；3g/L
Bacteriolyze enzyme solution react 7min；The bacteriolyze enzyme solution of 2g/L reacts 8min；The bacteriolyze enzyme solution of 1g/L reacts 10min.
Kit described in step (7) are as follows: the raw work RNA extracts kit in Shanghai, BioFlux RNA extracts kit,
One of TaKaRa RNA extracts kit, Hangzhou treasured match biology RNA extracts kit；Although different kit reagents are not
Together, but main agents are typically all lysate, protein liquid removal, rinsing liquid.
The needs of deproteinized step described in step (7) are repeated 2 times, and need to get rid of 1min in sky under 12000rpm after rinsing, then
Room temperature dries 5min.
RNA Concentration Testing described in step (8) needs 1 μ L, 9 μ L of residue that the loading buffer of 1 μ L is added to nucleic acid
Electrophoresis, nucleic acid electrophoresis applied sample amount are 2-6 μ L, and nucleic acid glue is prepared using the agarose of 2%-3%, and electrophoresis tank is slow when running glue
The TCA buffer that fliud flushing needs instantaneously changing new.
The RNA of 0.5 μ L in addition RT-PCR reaction system described in step (9), concentration range is in 0.2-1g/L；With
It need to be controlled with the cDNA concentration of RT-qPCR in 200 ± 50ng/ μ L.
The present invention is beneficial to be had the technical effect that
It is common to be suitable for kinds of experiments room for bacillus licheniformis RNA extraction method in fermentation process provided by the present invention
Antibacterial agents box (the raw work RNA extracts kit in Shanghai, BioFlux RNA extracts kit, TaKaRa RNA extracts kit,
Hangzhou treasured matches biology RNA extracts kit etc.), the method for the present invention can significantly improve bacillus licheniformis RNA and extract quality, and
And fermentation liquid needed for RNA extraction is less (50-300 μ L)；The RNA of extraction has purity is high, moderate concentration, stability height etc. excellent
Point can sufficiently meet the downstream applications such as RT-PCR, RT-qPCR.
Detailed description of the invention
Fig. 1 is the RNA electrophoretogram of different time points during the lichen bacillus ferments that the method for the present invention is extracted；
In figure: swimming lane 1-14 is respectively to ferment to 7,10,12,14,16,18,20,23,26,29,32,36,40,46h
Bacillus licheniformis RNA；Band is respectively 23S rRNA, 16S rRNA, 5S rRNA from top to bottom in swimming lane.
Fig. 2 is the portable RNA method of tradition and the RNA that laboratory is often extracted with e. coli rna isolation kit method respectively
Electrophoretogram；
In figure: applied sample amount is 2 μ L, is consistent with Fig. 1；Swimming lane 1 and 2 is the lichens that the portable RNA method of tradition is extracted
Bacillus RNA；Swimming lane 3 and 4 is the bacillus licheniformis RNA that laboratory often uses e. coli rna kit to extract.
Fig. 3 is the RNA electrophoretogram of the lichen bacillus ferments process under different temperatures；
In figure: applied sample amount is 2 μ L, is consistent with Fig. 1；Swimming lane 1 and 2 is respectively the RNA sample of 12h and 46h at 25 DEG C
Product；Swimming lane 3 and 4 is respectively the RNA sample of 12h and 46h at 30 DEG C；Swimming lane 5 and 6 is respectively the RNA sample of 12h and 46h at 37 DEG C
Product.
Fig. 4 is the RNA electrophoretogram of the lichen bacillus ferments process under different pH；
In figure: applied sample amount is 2 μ L, is consistent with Fig. 1；Swimming lane 1-4 is respectively pH 4.15, pH 5.72, pH
6.51, the RNA sample of pH 7.14.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
The extraction of different stages of growth bacillus licheniformis RNA and its RT-PCR, RT-qPCR
Fermentation liquid is shaken up in fermentation process, 500 μ L of real time sample is placed in 1.5mL Ep pipe, and 60 μ L bacterium solutions is taken to dilute
Fermentation liquid OD600 is measured after 50 times, remaining sample is immediately placed in -80 DEG C of refrigerators and saves；
After fermentation ends 2 days, the 14 bacteria liquid sample whole ice bath 0.5h saved in -80 DEG C of refrigerators are thawed in advance；
Take the bacterium solution after thawing as in 1.5mL Ep pipe, wherein the collected volume of fermentation 7-20h fermentation liquid is 2000/
OD600 μ L, the collected volume of fermentation 20-48h fermentation liquid are 800/OD600 μ L, and samples taken is centrifuged in room temperature 12000rpm
30s；
Supernatant is outwelled, 600 μ L ddH are added2O, on vortex mixer be resuspended thallus 30s, room temperature 12000rpm from
Heart 30s；
Supernatant is outwelled, 1mL ddH is added2O shifts the bacterium solution after dilution in thallus 30s is resuspended on vortex mixer
In the mortar crossed to Liquid nitrogen precooler, 600 μm of microglass bead and liquid nitrogen are added, is ground using the mortar of diameter 100mm
At powder, it is carefully transferred in 1.5mL Ep pipe with key immediately after, grinds 3 samples respectively using 3 mortars every time
The sample ground is placed in -80 DEG C of refrigerators and saved by product.
After above-mentioned sample ice bath thaws, the bacteriolyze enzyme solution of the 3g/L of 100 μ L Tris-HCl buffers, Yu Xuan is added
Thallus 30s is resuspended on the mixer of whirlpool, then enzyme digestion reaction 7min at room temperature；
By taking BioFlux kit as an example, according to the explanation that kit provides, lysate R1 and R2 are sequentially added, in transfer
For clear liquid into purification column, 12000rpm is centrifuged 30s, outwells waste liquid, sequentially adds cleaning solution and washs 2 times, at 12000rpm
Sky gets rid of 1min, and then room temperature dries 5min, is eventually adding the RNase-Free water elution of 30 μ L, collects RNA；
The RNA of collection is divided to for two pipes.Wherein a pipe fills 10 μ L, take wherein 1 μ L with micro ultraviolet specrophotometer to RNA into
Row Concentration Testing；After the loading buffer of 1 μ L is added in the RNA of 9 μ L of residue, 2 μ L is taken to carry out nucleic acid electrophoresis, nucleic acid glue uses
2% agarose is prepared, the buffer instantaneously changing of electrophoresis tank new TCA buffer when running glue.Another pipe fills the RNA of 20 μ L,
It is placed in -80 DEG C of refrigerators and is saved with liquid nitrogen frozen, remain RT-PCR.
When carrying out RT-PCR, takes the RNA of 0.5 μ L as template, reverse transcription enzyme system is added, cDNA is synthesized with this.Carry out RT-
When qPCR, using the Ribosomal protein S5 gene rpsE in bacillus licheniformis ATCC 9945a genome sequence as reference gene；
Using the xylose isomerase gene xylA of xylose induction as purpose gene, 0.5 μ L is taken to be diluted to the cDNA conduct of 200 ± 50ng/ μ L
Template terminates post analysis data.
The RNA electrophoretogram (Fig. 1) of the lichen bacillus ferments different time points, different growth steps are obtained according to the method
Bacterium solution volume needed for section bacillus licheniformis RNA is extracted and RNA evaluation parameter (table 1) and RT-PCR and RT-qPCR result ginseng
Number (table 2).
Table 1
Table 2
As seen from Figure 1, RNA purity is higher, and the brightness of 23S rRNA is greater than 16S rRNA, and the brightness of 5S rRNA is lower, nothing
Apparent degradation band, illustrates that total serum IgE integrality is preferable.Although the step in summary of the invention is increased in Fig. 2 when extracting RNA
Suddenly the bacteriolyze enzyme solution of the 3g/L of 100 μ L Tris-HCl buffers is added, in vortex mixer in (5) during the extraction process
Upper resuspension thallus 30s, then enzyme digestion reaction 7min at room temperature.But no matter uses the portable RNA method of tradition or use common
The RNA concentration that RNA isolation kit extracts is lower, and pollution is more serious, and containing more genomic DNA, this shows only to walk
Suddenly (5), lytic effect is not thorough, and every kind of method has limitation.In conjunction with table 1, A260/A280 between 2.1-2.2,
Illustrate that no albumen or phenolic substances influence；A260/A230 illustrates not have substantially carbohydrate, more also between 2.1-2.2
Peptides pollution, these results show that the bacillus licheniformis RNA purity extracted using the method for the invention is higher；And RNA
For concentration between 400-900, concentration is also more moderate.According to table 2, carrying out RT-PCR as template using the RNA of extraction can be obtained
Obtain the cDNA of higher concentration；And in RT-qPCR, the reference gene rpsE and mesh that are obtained using the cDNA of RNA reverse transcription as template
Gene xylA Ct value collimation it is fine, Ct value is between 15-30, and reference gene rpsE and target gene
The gene expression abundance of xylA is moderate, illustrates that the bacillus licheniformis RNA extracted according to the method for the invention is completely suitable for RT-
The downstream applications such as PCR and RT-qPCR.
Embodiment 2
The extraction of bacillus licheniformis RNA in different temperatures fermentation process
Fermentation liquid is shaken up in fermentation process, 500 μ L of real time sample is placed in 1.5mL Ep pipe, and 60 μ L bacterium solutions is taken to dilute
Fermentation liquid OD600 is measured after 50 times, remaining sample is immediately placed in -80 DEG C of refrigerators and saves；After fermentation ends 5 days, in advance
The 6 bacteria liquid sample whole ice bath 1h saved in -80 DEG C of refrigerators are thawed；Take the bacterium solution after thawing into 1.5mL Ep pipe, when
When fermentation liquid OD600 is less than 20, collected bacterium solution volume is 2000/OD600 μ L；It is collected when fermentation liquid OD600 is greater than 30
Bacterium solution volume is 800/OD600 μ L, and samples taken is centrifuged 30s in room temperature 12000rpm；
Supernatant is outwelled, 800 μ L ddH are added2O, on vortex mixer be resuspended thallus 30s, room temperature 12000rpm from
Heart 30s；
Supernatant is outwelled, 1mL ddH is added2O shifts the bacterium solution after dilution in thallus 30s is resuspended on vortex mixer
In the mortar crossed to Liquid nitrogen precooler, 425 μm of microglass bead and liquid nitrogen are added, is ground into using the mortar of diameter 60mm
It is carefully transferred in 1.5mL Ep pipe with key by powder immediately after, grinds 3 samples respectively using 3 mortars every time,
The sample ground is placed in -80 DEG C of refrigerators to save.
After above-mentioned sample ice bath thaws, the bacteriolyze enzyme solution of the 1g/L of 100 μ L Tris-HCl buffers, Yu Xuan is added
Thallus 30s is resuspended on the mixer of whirlpool, then enzyme digestion reaction 10min at room temperature；
By taking TaKaRa kit as an example, according to kit specification, lysate RL, dehydrated alcohol are sequentially added, in transfer
For clear liquid into purification column, 12000rpm is centrifuged 30s, outwells waste liquid, sequentially adds protein liquid removal RW1, rinsing liquid RW is washed respectively
It washs 2 times, sky gets rid of 1min at 12000rpm, and then room temperature dries 5min, is eventually adding the RNase-Free water elution of 50 μ L, receives
Collect RNA；
The RNA of collection is divided to for two pipes.Wherein a pipe fills 10 μ L, take wherein 1 μ L with micro ultraviolet specrophotometer to RNA into
Row Concentration Testing；After the loading buffer of 1 μ L is added in the RNA of 9 μ L of residue, 2 μ L is taken to carry out nucleic acid electrophoresis, nucleic acid glue uses
3% agarose is prepared, the buffer instantaneously changing of electrophoresis tank new TCA buffer when running glue.Another pipe fills the RNA of 20 μ L,
It is placed in -80 DEG C of refrigerators and is saved with liquid nitrogen frozen, it is spare.
The RNA electrophoretogram (Fig. 3) of the lichen bacillus ferments process under different temperatures, comparison diagram are obtained according to the method
1, it is seen that RNA extraction method of the present invention is stablized, and the RNA mass of extraction is preferable.
Embodiment 3
The extraction of bacillus licheniformis RNA in different pH fermentation process
Fermentation liquid is shaken up in fermentation process, 500 μ L of real time sample is placed in 1.5mL Ep pipe, and 60 μ L bacterium solutions is taken to dilute
Fermentation liquid OD600 is measured after 50 times, remaining sample is immediately placed in -80 DEG C of refrigerators and saves；After fermentation ends 3 days, in advance
The 4 bacteria liquid sample whole ice bath 0.8h saved in -80 DEG C of refrigerators are thawed；
Take the bacterium solution after thawing into 1.5mL Ep pipe, when fermentation liquid OD600 is less than 20, collected bacterium solution volume is
2000/OD600μL；When fermentation liquid OD600 is greater than 30, collected bacterium solution volume is 800/OD600 μ L, by samples taken in room
Warm 12000rpm is centrifuged 30s；
Supernatant is outwelled, 800 μ L ddH are added2O, on vortex mixer be resuspended thallus 30s, room temperature 12000rpm from
Heart 30s；
Supernatant is outwelled, 1mL ddH is added2O shifts the bacterium solution after dilution in thallus 30s is resuspended on vortex mixer
In the mortar crossed to Liquid nitrogen precooler, 500 μm of microglass bead and liquid nitrogen are added, is ground using the mortar of diameter 130mm
At powder, it is carefully transferred in 1.5mL Ep pipe with key immediately after, grinds 2 samples respectively using 2 mortars every time
The sample ground is placed in -80 DEG C of refrigerators and saved by product.
After above-mentioned sample ice bath thaws, the bacteriolyze enzyme solution of the 5g/L of 100 μ L Tris-HCl buffers, Yu Xuan is added
Thallus 30s is resuspended on the mixer of whirlpool, then enzyme digestion reaction 5min at room temperature；
By taking Hangzhou treasured matches biology RNA extracts kit as an example, according to kit specification, lysate BRL is sequentially added, it will
Upper layer RNA solution is transferred to the dehydrated alcohol that new pipe adds 1/2 times of volume, is transferred in purification column after mixing, 12000rpm
It is centrifuged 30s, outwells waste liquid, sequentially adds protein liquid removal RP, rinsing liquid RW is washed 2 times respectively, sky is got rid of at 12000rpm
1min, then room temperature dries 5min, is eventually adding the RNase-Free water elution of 40 μ L, collects RNA；
The RNA of collection is divided to for two pipes.Wherein a pipe fills 10 μ L, take wherein 1 μ L with micro ultraviolet specrophotometer to RNA into
Row Concentration Testing；After the loading buffer of 1 μ L is added in the RNA of 9 μ L of residue, 2 μ L is taken to carry out nucleic acid electrophoresis, nucleic acid glue uses
2% agarose is prepared, the buffer instantaneously changing of electrophoresis tank new TCA buffer when running glue.Another pipe fills the RNA of 20 μ L,
It is placed in -80 DEG C of refrigerators and is saved with liquid nitrogen frozen, it is spare.
Obtain the RNA electrophoretogram (Fig. 4) of the lichen bacillus ferments process under different pH according to the method, comparison diagram 1,
It can be seen that RNA extraction method of the present invention is stablized, the RNA mass of extraction is preferable.
In conjunction with above example, a kind of method that the bacillus licheniformis RNA suitable for fermentation process is extracted can be proposed, with
Meet the downstream applications demand such as RT-PCR and RT-qPCR, so as to be based on gene expression regulation principle to bacillus licheniformis
The further application of this essential industry microorganism points the direction.
The foregoing is merely illustrative of the preferred embodiments of the present invention, more at large describes the content of present invention, and
Not to limit the present invention.All within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done,
It should be included within protection scope of the present invention.
Claims (10)
1. a kind of extracting method of the bacillus licheniformis RNA suitable for fermentation process, which is characterized in that the extracting method packet
Include following steps:
(1) according to biomass, the fermentation liquid during the lichen bacillus ferments is collected；
(2) fermentation liquid obtained to step (1) is washed, is crushed, enzymolysis processing；
(3) using kit to by step (2) treated that sample cracked, clean, wash, elute.
2. extracting method according to claim 1, which is characterized in that the specific steps of the method are as follows:
(1) fermentation liquid is shaken up in fermentation process, 500 μ L of real time sample is placed in 1.5mL Ep pipe, and 60 μ L bacterium solutions is taken to dilute 50
Fermentation liquid OD600 is measured after times, remaining sample is immediately placed in -80 DEG C of refrigerators and saves；
(2) to which after fermentation, the bacterium solution ice bath 0.5-1h saved in -80 DEG C of refrigerators thaws in advance；
(3) according to the OD600 measured, the thalline quantity in samples taken is controlled in suitable range: (0.2-0.5) ×
109Cfu, that is, take thaw after bacterium solution (800-2000)/OD600 μ L in 1.5mL Ep pipe, respectively room temperature 12000rpm from
Heart 30s；
(4) supernatant is outwelled, 600-800 μ L ddH is added2O, in thallus 30s is resuspended on vortex mixer, in room temperature 12000rpm
It is centrifuged 30s；
(5) supernatant is outwelled, 1mL ddH is added2Bacterium solution after dilution is transferred to by O in thallus 30s is resuspended on vortex mixer
In the mortar that Liquid nitrogen precooler is crossed, microglass bead and liquid nitrogen are added, grind into powder is immediately after carefully shifted it with key
Into 1.5mL Ep pipe, the sample being disposed can place -80 DEG C of preservations.
(6) the bacteriolyze enzyme solution of the 1-5g/L of 100 μ L Tris-HCl buffers is added in step (5) treated sample, in
Thallus 30s is resuspended on vortex mixer, then enzyme digestion reaction 5-10min at room temperature；
(7) according to kit specification, lysate, protein liquid removal, rinsing liquid are sequentially added, then sky gets rid of, dries, and is eventually adding
The RNase-Free water elution of 30-50 μ L collects RNA；
(8) RNA of 10 μ L is taken to be used to carry out Concentration Testing, nucleic acid electrophoresis, remaining RNA is placed on -80 DEG C of ice with liquid nitrogen frozen
It is saved in case；
(9) it when carrying out a RT-PCR or step RT-qPCR, takes the RNA of 0.5 μ L as template, reverse transcription enzyme system or qPCR enzyme is added
System, synthesizes cDNA with this and is reacted.
3. extracting method according to claim 2, which is characterized in that fermentation liquid described in step (1) is in -80 DEG C of refrigerators
The preservation time must not exceed 5 days.
4. extracting method according to claim 2, which is characterized in that thalline quantity described in step (3) and OD600's
Relational expression: calculating according to 0.25 × 109cfu/mL of 1OD600 ≈, when fermentation liquid OD600 is less than 20 in fermentation process,
Collected bacterium solution volume is 2000/OD600 μ L；When fermentation liquid OD600 is greater than 30 in fermentation process, collected bacterium solution volume is
800/OD600μL。
5. extracting method according to claim 2, which is characterized in that microglass bead diameter described in step (5) is 425-
600 μm, mortar diameter is 60mm, 80mm, 90mm, 100mm or 130mm.
6. extracting method according to claim 2, which is characterized in that concentration described in step (5) is the lysozyme of 5g/L
Liquid reacts 5min；The bacteriolyze enzyme solution of 4g/L reacts 6min；The bacteriolyze enzyme solution of 3g/L reacts 7min；The bacteriolyze enzyme solution of 2g/L reacts
8min；The bacteriolyze enzyme solution of 1g/L reacts 10min.
7. extracting method according to claim 2, which is characterized in that kit described in step (7) are as follows: the raw work in Shanghai
RNA extracts kit, BioFlux RNA extracts kit, TaKaRa RNA extracts kit, Hangzhou treasured match biology RNA are extracted
One of kit；Although different kit reagents are different, main agents are typically all lysate, protein liquid removal, rinsing
Liquid.
8. extracting method according to claim 2, which is characterized in that deproteinized step described in step (7) needs to repeat 2
It is secondary, 1min need to be got rid of in sky under 12000rpm after rinsing, then room temperature dries 5min.
9. extracting method according to claim 2, which is characterized in that RNA Concentration Testing described in step (8) needs 1 μ L, remains
The loading buffer of 1 μ L is added to nucleic acid electrophoresis in 9 μ L of remaininging, and nucleic acid electrophoresis applied sample amount is 2-6 μ L, and nucleic acid glue uses
The agarose of 2%-3% is prepared, the TCA buffer that the buffer of electrophoresis tank needs instantaneously changing new when running glue.
10. extracting method according to claim 2, which is characterized in that RT-PCR reaction system is added described in step (9)
In 0.5 μ L RNA, concentration range is in 0.2-1g/L；CDNA concentration to RT-qPCR need to be controlled in 200 ± 50ng/ μ
L。
Priority Applications (1)
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