BM Seamless Cloning Kit (Biomed, Beijing, China), DH5 α and Agrobacterium GV3101(Biomed, Beijing, China), small plasmid extraction kit (TIANGEN, Sichuan, China), Gel recovery, DL2000 Marker, High fidelity enzyme and LA Taq (TaKaRa, Beijing, China), X-gal and IPTG (Real Times, Shenzhen, China), Bis, Tris Hcl, SDS, TEMED, ammonium persulfate, ammonium persulfate, kana, sucrose, and agar powder (Sinopharm, Beijing, China), and LB medium (ShengGong, Liaoning, China).
Plasmid small extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The Plasmid Small Extraction Kit is a laboratory tool designed for the rapid and efficient extraction of plasmid DNA from bacterial cultures. It utilizes a series of buffers and centrifugation steps to isolate plasmid DNA from small-scale bacterial preparations.
Automatically generated - may contain errors
Lab products found in correlation
Dl2000 marker, by Takara Bio (1 mentions)
Sodium dodecyl sulfate (sds), by Sinopharm (1 mentions)
Ammonium persulfate, by Sinopharm (1 mentions)
Tris hcl, by Sinopharm (1 mentions)
Engen lba cas12a, by New England Biolabs (1 mentions)
Premix taq, by Takara Bio (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Tcs sp8, by Leica (1 mentions)
6 protocols using plasmid small extraction kit
1
Seamless Cloning Protocol for Plasmids
2
Engineered SM22 Gene Delivery via AAV9
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The target genes(EnSM22 -mStk11-P2A-EGFP and EnSM22 .EGFP) and adeno-associated virus 9(AAVs9) were purchased from Saiye Biotechnology Co., Ltd. All target genes were knocked into EnSM22 , a VSMCs-speci c promoter. The circular plasmid expression vector was digested in water bath at 37 ℃, and the large fragments of the digested plasmid were recovered by 1% agarose gel electrophoresis. The large fragment and the target gene CDS sequence were recovered for gene recombination, and the recombination reaction was carried out using a seamless cloning kit (Novozan Biotechnology Co., Ltd., China). The plasmid was extracted with a small plasmid extraction kit (Tiangen Biotechnology Co., Ltd.), the plasmid was identi ed by enzyme digestion, and the positive clones were identi ed by sequencing. The correct recombinant plasmid was used in the next step of cell transfection.
The target plasmid and two helper plasmids were co-transfected into HET293T Cells for 72 hours. The supernatant was concentrated with PEG8000, and the virus was collected by repeated freeze-thaw lysis.
After puri cation and concentration, the titer of the virus was≥1E+13GC/mL determined by quantitative polymerase chain reaction.
The target plasmid and two helper plasmids were co-transfected into HET293T Cells for 72 hours. The supernatant was concentrated with PEG8000, and the virus was collected by repeated freeze-thaw lysis.
After puri cation and concentration, the titer of the virus was≥1E+13GC/mL determined by quantitative polymerase chain reaction.
3
Cloning and Purification of ASFV B646L
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The B646L gene of ASFV genotype II strain was synthesized by Zhejiang Sunya Biotechnology Co., Ltd China according to a published sequence (GenBank: MK333180.1 ), and cloned into the pUC57 to obtain the plasmid pUC57-B646L. The E.coli JM109 containing the recombinant plasmid was cultured and the plasmid DNA was extracted (Tiangen, Plasmid Small Extraction Kit, DP103-02) for further study. The viral nucleic acid of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine circovirus type 4 (PCV4), porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV) were purified and stored at the Key Laboratory of Animal Virology, Ministry of Agriculture, Zhejiang University.
4
Synthesis and Characterization of MRSA mecA Gene
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Partial sequences of MRSA mecA gene were synthesized by Sangon Biotech and cloned into pUC57 vectors, and transformed into E. coli DH5 . Recombinant pUC57 plasmid DNA containing the mecA gene was extracted by Plasmid Small Extraction Kit (Tiangen, Beijing, China). Then, we used M13F and M13R as primers and the extracted plasmid DNA as a template to amplify the mecA gene on Biometra Tone-PCR Thermal Cycler (Analytik Jena, Jena, Germany.) The PCR procedure follows the instructions of Premix Taq (Takara Bio Inc., Beijing, China): 30 cycles by 98 C for 10 s, followed by 52 C for the 30 s, and 72 C for 1 min. Finally, the PCR products were analyzed by 1% agarose gel electrophoresis. The Cas12a fluorescence detection experiment was carried out using EnGen® LbaCas12a (NEB, Ipswich, MA, USA) and above purified CrRNAs. For each reaction, 1 L of LbaCas12a (1 M) preassembled with 100 ng of CrRNA and 0.25 L RNA inhibitor. Then, 1 L ssDNA FQ Reporter and 1.5 L of 10× NEBuffer 2.1 were mixed; finally, 3 L above PCR products were added and immediately incubated at 42 C on a LightCycler 96 system (Roche, Basel, Switzerland) to measure the Fluorescence kinetics.
5
TPI1 Gene Silencing and Overexpression
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The TPI1-siRNA interference sequence was designed according to our earlier cloned Tibetan sheep TPI1 gene sequence (MN847717), and the negative control (NC-siRNA) was used as the control group (S1 Table ). Notably, both the TPI1-siRNA and NC-siRNA vectors were synthesized by Genepharma Biological. On the other hand, the TPI1 overexpression vector (pc-DNA-3.1(+)-TPI1) was constructed by Genewiz Biological, and the empty vector (pc-DNA-3.1(+) was used as the control group. The plasmid small extraction kit (Tiangen, Beijing, China) was used to extract the plasmid according to the manufacturer’s instructions, and then the recombinant plasmid was double digested with NheI and NotI restriction enzymes. Finally, the digestion products were identified by 2% agarose gel electrophoresis.
6
VvMYBPA1 Overexpression in Tobacco
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The VvMYBPA1 CDs without a terminator was amplified using the cNDA of young fruit of ‘Zaohaibao’ grape as the template, and the digestion site sequences were added in the primers (Supplementary Table S1 ), finally obtaining 35S::VvMYBPA1-GFP fusion vector. The VvMYBPA1 amplified product was connected to pMD-19T (Japan TaKaRa Bio Company, Osaka, Japan) and was transferred into E. coli Trans 5α in the competent state (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China), and the plasmid was extracted with the plasmid small extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Beijing, China) and sequencing. The plasmid and pCAMBIA1300 empty vector was digested with restriction endonuclease (Japan TaKaRa Bio Co., Ltd., Osaka, Japan), and connected with T4 ligase (Japan TaKaRa Bio Co., Ltd., Osaka, Japan), obtaining the 35S::VvMYBPA1-GFP plant expression vector. The fusion expression vector 35S::VvMYBPA1-GFP was transformed into the competent state of Agrobacterium tumefaciens GV3101 (Shanxi Shuoke Biotechnology Co., Ltd., Xi’an, China), and infected four weeks old tobacco leaves. Fluorescence signals were detected using laser confocal microscope (Leica TCS SP8, Leica, Wetzlar, Germany) after two/three days.
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