The coding sequence (CDS) of ROP18 of T. gondii RH strain (GenBank No. JX045330) was amplified from total RNA extracted from tachyzoite of T. gondii RH strain using the primers: ROP18-F (5’-GGGGGATCCATGACACTTGGTCCTTCAAAACTCG-3’) and ROP18-R (5’-GGGGTCGACTTCTGTGTGGAGATGTTCCTGCTGTTC-3’). The PCR conditions were set as follows: pre-denaturation for 5 min at 98°C followed by 35 cycles of 98°C for 20 s, 56°C for 18 s, and 72°C for 30 s; 72°C for 5 min and hold at 4°C. The PCR product was purified using Gel Extraction kit (OMEGA, China). The purified ROP18 CDS was cloned into PCMV-N-HA vector using BamHI and SalI restriction enzymes (NEB, USA), according to the manufacturer’s instructions. The constructed plasmid (PCMV-N-HA-ROP18) was transformed into E. coli DH5α competent cells (TIANGEN, China). Single bacterial colony was randomly selected and identified using PCR primers ROP18-F and ROP18-R. Positive colonies were sequenced by Genscript Corporation (Nanjing, China). The plasmid of PCMV-N-HA-ROP18 bacterial colony was extracted using Endofree Plasmid Kit (TIANGEN, China) following the manufacturer’s instructions, and the extracted plasmid was stored at −20°C until use.
E coli dh5α competent cells
Manufactured by Tiangen Biotech
Sourced in China
E. coli DH5α competent cells are a widely used laboratory strain of Escherichia coli bacteria. They are genetically engineered to be highly efficient at DNA uptake and recombination, making them a common choice for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Bl21 de3, by Tiangen Biotech (2 mentions)
Bamhi and sali restriction enzymes, by New England Biolabs (1 mentions)
Endofree plasmid kit, by Tiangen Biotech (1 mentions)
Xylan, by Yuanye Bio-Technology (1 mentions)
Microcrystalline cellulose, by Yuanye Bio-Technology (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Chitosan, by Yuanye Bio-Technology (1 mentions)
Gel extraction kit, by Tiangen Biotech (1 mentions)
Tamibarotene, by Merck Group (1 mentions)
Pgl3.0 basic, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Prl sv40, by Promega (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Dna ladder marker, by Takara Bio (1 mentions)
Pfu dna polymerase, by Promega (1 mentions)
Restriction endonucleases nde 1, by Thermo Fisher Scientific (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Premix la taq, by Takara Bio (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
7 protocols using e coli dh5α competent cells
1
Cloning and Expression of T. gondii ROP18
2
Constructing BPHA Plasmid for AGRxPD1 BsAb
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18A4HU plasmid (for AGR2 mAb) was constructed previously in our lab (Guo et al 2016 (link)), and that plasmid was used to construct BPHA (for AGRxPD1 BsAb) plasmid. The anti-PD1 sequence was constructed and joined with 18A4HU antibody sequence with a linker under EF-1a promoter, which was performed by overlap extension PCR with a DNA polymerase kit (KOD, Tyobo, China). The AmpR promoter controlled the ampicillin resistance (AmpR) coding sequence. Puromycin resistance (PuroR) coding sequence and EGFP coding sequence were both controlled by the same Cytomegalovirus (CMV) promoter. Both coding sequence is for the expression in mammalian cells. The puromycin coding sequence is for the selecting and generating of the stable cell line. Vector and all sequences were designed and constructed by using snapgene software. The newly constructed BPHA plasmid was transformed into E. coli (DH5α) competent cells (Tiangen Biotech, Beijing, #CB101). All constructs were analyzed and confirmed by DNA sequencing.
3
Enzymatic Deconstruction of Chitin Oligomers
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T. gamsii R1 was isolated from shrimp shell waste and identified via ITS sequencing and then conserved in our laboratory. E. coli Dh5α competent cells were purchased from Tiangen Biotechnology (Beijing, China). PrimeSTAR® HS (Premix) DNA polymerase and PrimeScript™ RT-PCR Kits were purchased from Takara Biotechnology (Beijing, China). COSs with different degrees of polymerization (DPs), including N-acetyl-D-glucosamine (DP1), N-acetyl-chitobiose (DP2), N-acetyl-chitotriose (DP3), N-acetyl-chitotetraose (DP4), and N-acetyl-chitopentaose (DP5), were purchased from Qingdao BZ Oligo Biotech (Qingdao, China). Furthermore, GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5 represent N-acetyl-D-glucosamine, N-acetyl-chitobiose, N-acetyl-chitotriose, N-acetyl-chitotetraose, and N-acetyl-chitopentaose, respectively. Powdery chitin, xylan, microcrystalline cellulose, and chitosan with a degree of deacetylation (DDA) of 85 to 95% were purchased from Yuanye Biotechnology (Shanghai, China). The colloidal and ball milled-chitin were prepared according to a previously described method [51 (link)].
4
Constructing pEGFP-N1 Vector in DH5α Cells
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The pEGFP-N1 vector was constructed in our laboratory. E. coli DH5α competent cells, gel extraction kit, and miniprep kits were purchased from the Tiangen Company (Beijing, China). DL5000 DNA Marker Prime STARMax DNA Polymerase, T4 DNA ligase, and restriction endonuclease were purchased from Takara (Dalian, China). The expression vector pGL3.0-Basic, pRL-SV40, and the Dual-Luciferase Reporter Assay System were purchased from Promega Corporation. LipofectamineTM2000 was purchased from Invitrogen Corporation. ATRA, Tamibarotene (Tamibarotene, Am80), and TSA were purchased from SIGMA Company. The chicken embryo fibroblast cell line, DF-1, and Mouse spermatogonial cell line, GC-1,were purchased from ATCC. Primer synthesis and sequencing were conducted by the Invitrogen Company of Shanghai (China).
5
E. coli Strain Cultivation and Plasmid Transformation
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E. coli strain K12 (MG 1655) purchased from the American Type Culture Collection (ATCC 15597) was grown in lysogeny broth (LB) (10 g/L tryptone (Difco), 5 g/L yeast extract (Difco), 10 g/L NaCl) at 37°C. E. coli DH5α competent cells purchased from TIANGEN BIOTECH (Beijing, China) were cultured in LB at 37°C. E. coli with pRedCas9 plasmid was cultured in LB at 30°C. Table S15 lists the bacteria strains and plasmids used in this study. Table S16 lists the primers used in this study.
6
Recombinant hEGF Production Protocol
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Competent E. coli cells DH5α and BL21(DE3) were purchased from TIANGEN Biotech (Beijing, China). Plasmids pTWIN1, pET28a-SUMO, and pET21a were preserved by our own laboratory. Restriction endonucleases Nde I and Xho I were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Prime STAR HS (Premix) LA Taq, DNA ladder Marker, and kits for DNA manipulation were purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China). Protein ladder marker was obtained from Thermo Fisher Scientific (CA, USA). Pfu DNA polymerase was purchased from Promega (Madison, USA). hEGF coding region was synthesized by Huada Genomics Institute Co., Ltd. (Shenzhen, China). Commercial recombinant hEGF was purchased from Peprotech Co., Ltd. (Rocky Hill, USA). Mouse fibroblast Balb/c 3T3 cells were kindly given by Professor Yadong Huang from Jinan University. All chemicals used in this study were of analytical grade.
7
Recombinant Protein Expression in E. coli
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Competent E. coli cells DH5α and BL21 (DE3) were purchased from TIANGEN Biotech (Beijing, China). Restriction endonucleases NdeI and XhoI, Premix LA Taq, and DNA Ligation Kit were purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China). SP2/0-Ag14 myeloma cells and pET-28a plasmid were preserved in our lab. All chemicals used in this study were of analytical grade.
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