1) A tube of 100 μL DH5α was inoculated in 5 mL LB liquid medium and incubated overnight in shaker at 37 ℃, 160 rpm/min.

2) Inoculated the overnight culture in 100 mL of LB medium at 1% ratio and incubated at 37 ℃ for 2-3 h (Cultured to OD

₆₀₀

=0.5-0.6).

3) Cooled the bacteria culture in the mixture of ice and water for 5-10 minutes in advance. Then transferred the treated bacteria culture to a centrifugal tube and placed it on ice for 10 mins to cool the bacteria liquid.

4) Transferred the cold bacterial liquid into 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins. Discarded the liquid supernatant and collected the bacterium.

5) Added 60 mL cold 0.1 mol/L CaCl

₂

solution to re-suspend the bacterium, and then cooled it in an ice bath for 45 mins.

6) Transferred the bacterial liquid into a 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins to discard the liquid supernatant and collected the bacterium.

7) Added 1 to 2 mL cold 15% glycerol solution (containing 0.1 mol/L CaCl

₂

solution) to re-suspend the bacterium, and divided them into 300 μL of each micro centrifuge tube and then stored at -80 ℃.

1) Added an appropriate amount of DNA sample or ligated reaction product to 100 μL competent cells, mixed slightly, and took an ice bath for 30 mins.

2) The micro centrifuge system was placed in a water bath at 42 ℃ for 90 s incubation.

3) After incubation, placed the competent cells quickly in the mixture of ice and water for cooling 2 mins, and then added into 800 μL LB liquid medium for shaking culture at 37 ℃ for 45-60 mins.

4) Took out, centrifuged at a speed of 8000 rpm/min for 1 min, removed the liquid supernatant, and left 100 μL.

5) Coated on LB solid plate containing corresponding antibiotics and cultured upside down overnight in 37 ℃ incubator to observe the growth of transformation.

1) Alkali lysate I, weighed 3.03 g Tris-base, 2.92 g EDTA, 9.9 g glucose into 1 L H

₂O, adjusted pH to 8.0 , 121 ℃ sterilized for 10 mins then stored at 4 ℃.

2) Alkali lysate II, mixed 0.4 mol/L NaOH and 2% SDS in equal volume, while 0.4 mol/L NaOH and 2% were prepared by sterile water respectively and stored at room temperature.

3) Alkali lysate III, weighed 194.42 g potassium acetate into 800 mL steriled water, then 115 mL of acetic acid was added, and finally the volume was fixed to 1 L, stored at 4 ℃.

4) 50% glycerol, equal volume of 100% glycerol and sterile water were mixed and stored at 4 ℃.

5) 2×GC buffer: added BSA 40 mg, Betaine 24.6 g, Tris-HCl 4 mL at pH 8.8, 1 mol/L KCl 4 mL, 800 μL of 1 mol/L Mg

₂

SO

₄

at pH 8.8, 12 mL of 0.5 mol/L (NH

₄ )₂SO₄

at pH 8.8, 24 mL 50% glycerin, 4 mL DMSO, 4 mL N, N, N-dimethylformamide, Triton X-100 400 μL, and finally the volume was fixed to 200 mL with sterilized water.

6) SPI salts, weighed (NH

₄ )₂

SO

₄

0.2 g, K

₂

HPO

₄

1.4 g, KH

₂

PO

₄

0.6 g, Na

₃

C

₆

H

₅

O

₇

·2 H

₂O, Mg

₂SO ₄·7 H₂O 0.02 g and finally the volume was fixed to 100 mL with ddH₂O, sterilized at 121 ℃ for 20 mins, and stored at room temperature.

7) Casein amino acid/yeast extract, weighed Casein amino acid 0.2 g, yeast extract 1 g , then the volume was fixed to 10 mL with ddH

₂O, then filtered and sterilized, and stored at room temperature after completion.

9) 250 mM MgCl

₂,

weighed MgCl

₂·6H₂O 0.5075 g and dissolved it in 20 mL ddH2O, and sterilized it at 121 ℃ for 20 mins, then stored at room temperature.

10) 100 mM EGTA, weighed ethyleneglycol-bis-(2-aminoethylether)-N,N'-tetraacisicacid (EGTA) 0.38 g and dissolved it in 10 mL ddH

₂

10)O, and adjusted pH to 7 with NaOH then filtered and sterilized, finally stored at room temperature after completion.

All reagent below referent was offered by Extract kit.

1) Added twice the volume of Binding Buffer to the sample, stood at room temperature for 1 min. Transferred to adsorption column, then centrifuged at a speed of 13000 rcf/min for 1 min and discarded the liquid supernatant of collect column (If the sample was attained from the gel extraction, the volume of Binding Buffer was exceeded the gel).

2) Added 300 μL Binding Buffer to adsorption column, then centrifuged at a speed of 10000 rcf/min for 1min and discarded the liquid supernatant of collect column.

3) Added 700 μL Washing Buffer to adsorption column, stood at room temperature for 2 mins, then centrifuged at a speed of 10000 rcf/min for 1 min and discarded the liquid supernatant of collect column (Repeated this step twice).

4) Added nothing to adsorption column, stood at room temperature for about 5 mins, then discarded collect column and transferred adsorption column to a new centrifuge tube.

5) Added 30~50 μL 55℃ ddH

₂O, stood at room temperature for 1 min and then centrifuged at a speed of 13000 rcf/min for 1 min.

6) Transferred the water in the centrifuge tube to adsorption column and then centrifuged one more time.

7) Discarded the adsorption column, and restored the sample at -20 ℃.

1) Sample Preparation, added electrophoretic Loading buffer, Gel Loading Dye, Purple(6×) into the products of endonuclease digestion, that was, added 1part buffer to 5 parts of sample.

3) Electrophoresis, completely immersed the agarose gel in the buffer, added mark and the sample into the loading well, and performed electrophoresis at 100 V for 45 mins.

4) Stain, placed the gel in 10 μg/mL EB dye solution for 2-4 mins at room temperature.

5) Observation and cutting, observed the electrophoresis tape and its position on the ultraviolet fluoroscope, and cut the fragments in the required range.

1) Weighed NaCl 10 g, Tryptone 10 g, Yeast extract 5 g then the volume was fixed to 1 L with ddH

₂O and adjusted pH to 7.0 with NaOH, sterilized at 121 ℃ for 20 mins, and stored at room temperature.

2) For solid LB medium, 1.5 g Agar powder was added every 100 mL on the basis of (1).

1) For the liquid medium, the corresponding antibiotics were added at a ratio of 1/1000 before using.

2) For the solid medium, it was heated and melted before using. When the temperature was reduced to about 50-60 ℃, the corresponding antibiotics were added in the proportion of 1/1000, which can be used to make the plate.

1) Took out a tube of chemocompetent cells, melted them on ice and added about 1 μg of DNA.

2) Incubated in shaker at 37 ℃, 160 rpm for 90 mins.

3) Added 300 μL LB medium and then incubated in shaker at 37 ℃, 160 rpm for 40 mins.

4) Took 50 to 100 μL to coat on LB solid plate containing antibiotics and cultured upside down overnight in 37 ℃ incubator to observe the growth of transformation.

1) Preparation 1-reaction PCR mix into the PCR tube by adding together 8.5 µL deionized water, 12.5 µL 2×GC buffer, 2 µL dNTP mix,1 μL templated DNA, 1 μL primer pairs and 0.125 µL rTaq DNA polymerase.

2) Placed the PCR tubes into the PCR apparatus and run it (Protocol can be divided into five centrifuge tubes, pre-denaturation → denaturation → anneal→ extension → final extension). After the PCR program entered denaturation, it run 30 times successively through denaturation, annealed and extension, and finally entered complete extension. After completing extension, waited for the temperature to drop to 16 ℃ to end the pro-gram.

① Pre-denaturation, run at 95 ℃ for 5-10 mins to completely denature the primes, so as to ensure that the primers were single stranded. Meanwhile, some secondary structures of the template DNA aredestroyed, and the double stranded of the template DNA was fully dissociated.

③ Anneal, reduced the solution temperature to the appropriate temperature (determined by annealing temperature pf the primer) and centrifuged it for 20 s to make the template DNA and primer complemented each other according to the principle of base pairing principle.

④ Extension, when the reaction temperature of the solution rose to 72 ℃, the rTaq DNA polymerase took the single strand DNA as the template and centrifuged the complementary DNA in the direction of 5'- 3' by using four kinds of deoxynucleoside triphosphate (dNTP) in the reaction mixture under the guidance of primers. The extension time dmicrocentrigugeended on the length of the fragment. The PCR instrument used in this experiment was extended by 1 kb/min.

⑤ Final extension, maintained it at 72 ℃ for 8 mins to make PCR reaction more completed to improve amplification yield.

⑥ Waited for the temperature to drop to 16 ℃ and the PCR procedure was completed.

1)All reagent below referent was offered by EASY spin Plus.

2)Extract RNA, ① Centrifuged the strains at a speed of 8000 rpm/min for 2 mins, and discarded the liquid supernatant. ② Added moderate lysozyme(about 100~200 μL) and cultured in 37 ℃ incubator for 30 mins. During the period, turned upside down and mixed for every five minutes. ③ Added 500 μL RLT Plus lysate and mixed sufficiently, shook violently for 20 s. ④ Added the lysate to DNA clear column, then centrifuged at 1300 rpm for 30 s, and retained the filtrates. ⑤ Added 500 μL 70% ethanol solution to the filtrates. ⑥ Added the mixture to the absorption column and then centrifuged at 13000 rpm for 30 s. ⑦ Added 700 μL free protein serum, stood at room temperature for 30s and then centrifuged at 13000 rpm for 30 s. ⑧ Added 500 μL rinse and centrifuged at 13000 rpm for 30 s twice. ⑨ Centrifuged without anything at 13000 rpm for 2 mins. ⑩ Transfer absorption column to centrifuge tube which has been heated at 121 ℃ for one hour. And then added 30~50 μL RNase-free H

₂O, stood at room temperature for 1 min and centrifuged at 13000 rpm for 1 min. Took out 5 μL for electrophoresis and measuring the concentration quickly, 8 μL for reverse cDNA and the others for storage.

3) Prepare the sample with DNaseⅠsystem (10 μL), 80%RNA, 10% DNaseⅠ, 10% 10× Buffer. Stood system at 37 ℃ for 30mins and 65 ℃ for 10 mins, then terminated reaction and stored on ice.

4) Reverse cDNA, Different RNA are adjusted by volume and concentration to the same amountof material. System, RNA, 500 ng, Random Primer, 1 μL, 5× TRUE Reaction Mix, 4 μL, added RNase-free H

₂ O to 20 μL. Every type RNA prepared 4 PCR tubes, each PCR tube contained 40 μL. Stood the system at 42 ℃ for 15 mins and at 85 ℃ for 5 s.

1) Single Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme, 5% ddH

₂O.

2) Double Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme A, 5% enzyme B.

3) Single enzyme digestion system of gene fragment, 80% plasmid, 10% buffer, 2% enzyme, 8% ddH

₂O.

4) Double enzyme digestion system of gene fragment, 80% gene fragment, 16% buffer, 2% enzyme A, 2% enzyme B.

1) Enzyme-linked system 1 (total volume 10 μL), 20% plasmid, 60% gene fragment, 10% buffer, 10% T4 DNA ligase.

2) Enzyme-linked system 2 (total volume 10 μL), 40% plasmid, 40% gene fragment, 10% buffer, 10% T4 DNA ligase.

3) Reaction condition, stood at 16 ℃ for 4 hours or overnight.