For RNAi experiments in A. thaliana, a 277-nt Miatrr fragment was amplified from cDNA of M. incognita with two-pair primers using Q5® High-Fidelity DNA Polymerases (M0419, NEB, MA, USA), and then inserted upstream and downstream of the pSAT5 intron in the forward and reverse orientations [34 (link)]. Then, after digestion with restriction enzymes XbaI and KpnI, the hairpin fragment was acquired and inserted into the pSUPER destination vector to construct the pSUPER-Miatrr-RNAi vector. Via Agrobacterium-mediated transformation, the RNAi vector was then transformed to A. thaliana Col-0 (wild-type) according to the floral dip method, following the description in a previous study [35 (link)]. Lines were verified via PCR and semiquantitative RT-PCR after hygromycin screening. Homozygous T3 plants from three Miatrr-RNAi lines were used for RNAi effect assay. The homozygous GFP-RNAi T3 lines used as control were the same as described in previous study [36 (link)]. The primers (synthesized by Tsingke Biotechnology), restriction enzymes (NEB, Beverly, MA, USA), and T4 ligase enzyme (M0202, NEB, Beverly, MA, USA) used for plasmid construction are listed in Supplementary Table S3 .
T4 ligase enzyme
Manufactured by New England Biolabs
Sourced in United States
T4 ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3' hydroxyl and 5' phosphate termini in DNA. It is commonly used in molecular biology applications such as DNA cloning and recombination.
Automatically generated - may contain errors
Lab products found in correlation
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Taq dna polymerase, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for qpcr, by Transgene (1 mentions)
Jm109 de3, by Promega (1 mentions)
Dna clean up kit, by CWBIO (1 mentions)
Fastpure plasmid mini kit, by Vazyme (1 mentions)
Xbai hf, by New England Biolabs (1 mentions)
Cell rna kit, by Yeasen (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Ecori hf, by New England Biolabs (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Bl21 de3, by Transgene (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Hieff trans liposomal transfection reagent, by Yeasen (1 mentions)
Bca protein quantification kit, by Yeasen (1 mentions)
Dh5alpha cells, by Thermo Fisher Scientific (1 mentions)
T4 ligation buffer, by New England Biolabs (1 mentions)
Stbl3 competent e coli cells, by Thermo Fisher Scientific (1 mentions)
Bbsi enzyme, by New England Biolabs (1 mentions)
6 protocols using t4 ligase enzyme
1
RNAi-mediated gene silencing in Arabidopsis
2
Molecular Cloning and Cell Culture Protocol
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High fidelity restriction endonucleases BamHI-HF, HindIII-HF, EcoRI-HF, XbaI-HF, and T4 ligase enzyme were purchased from NEB (New England Biolabs, MA, USA). Bacteria strain DH10B, BL21(DE3), Taq DNA polymerase, and All-in-One First-Strand cDNA Synthesis SuperMix for qPCR were purchased from Transgen, and JM109(DE3) was purchased from Promega (Promega, WI, USA). pT7Oi and pT7Om vectors were already constructed by our group. psilence-2.1-U6-Hygro vector was purchased from Beijing Rambolide Trading Co. DNA fragments were purified with AxyPrep DNA Gel Extraction Kit (Axygen) or DNA Clean-up Kit (Cwbio), and plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme) or Endo-Free Plasmid Mini Kit I (Omega). Cell RNA Kit, BCA protein quantification kit, and Hieff Trans liposomal transfection reagent were purchased from Yeasen. ProtLytic Protein Lysis and Sample Loading was purchased from New Cell & Molecular biotech Co. Human embryonic kidney 293T (HEK293T) was a gift from Professor Yao Shaohua’s laboratory. Michigan Cancer Foundation-7 (MCF-7) and human hepatocellular carcinomas (Hep G2) were purchased from ATCC (American Type Culture Collection, VA, USA).
3
Plasmid Construction for Pgc1α 3'UTR Study
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Full-length Pgc1α plasmid, GFP, and RFP reporter plasmids were purchased from Addgene (cat. #4, #35625, and #35626). Approximately 165 bp of mouse Pgc1α 3′UTR was synthesized from brown adipose tissue cDNA through PCR using AccuPrime Taq DNA polymerase and cloned into Pgc1α (Pgc1α-3′UTR), GFP (GFP-PGC1-3′UTR), and GFP (GFP-PGC1-3′UTRmt (mutation of two nucleotides in positions 4 and 5 at the seed region)) reporter plasmids with T4 ligase enzyme according to the manufacturer's instructions (New England Biolabs). Pgc1α 3′UTR was synthesized with a mutated site for miR-696 interaction using AccuPrime Taq DNA polymerase and cloned into GFP (GFP-PGC1-3′UTRmt) reporter plasmid. The 3′ and 5′ miR-696 oligonucleotide sequences (Eurofins) with BspQ1 overhangs were annealed for 5 min at 95 °C, followed by 10 min at 4 °C. Then the annealing product was cloned into an RFP reporter (RFP-miR696) plasmid using a BspQ1 enzyme (New England Biolabs). SNARK plasmids were generated as previously described [29 ]. Constructs were sequenced by the DNA Sequencing Core Facility at Brigham and Women's Hospital.
4
Bacterial Transformation and Plasmid Isolation
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The restriction enzymes, polymerase, and T4 ligase enzyme used for cloning and ligation were obtained from NEB. QIAGEN Plasmid isolation, gel extraction and PCR purification kits were used. For transformation, either NEB-5alpha competent E. coli (catalog #C2987H) or competent DH5alpha cells (originally obtained from Life Technologies) were prepared using the standard CaCl2 method of competent cell preparation. Bacterial culture media and agar (BD Biosciences) were prepared according to manufacturer’s instructions. Primers for the experiments were designed using A Plasmid Editor (Ape–version 1.17) and synthesized from Sigma. The primers received were diluted into stocks of 100 pmol/μL. The plasmid was sequenced by Genewiz.
5
Cloning gRNA into pKLV2 CRISPR Vector
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pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (pKLV2) was a gift from Kosuke Yusa (Addgene plasmid # 67974). Specific gRNA for EGFP and RIG-I (Table 2 , underlined) were cloned according to Golden Gate reaction from ZhangLab protocol (Addgene SAM library sgRNA cloning protocol) using BbsI-HF (NEB) and Stbl3 competent E. coli cells (Thermo Fisher) for transformation. Briefly, 1 μL of each oligo (100 μM) were mixed with 1 μL of T4 ligation buffer (NEB) and 7 μL of water. The mix was heated to 95 °C for 5 min and cooled to room temperature. 1 μL of the annealed oligos was mixed with 25 ng of pKLV2, 1 μL of T4 ligation buffer, 9 μL of water and 0.5 μL of BbsI enzyme and 0.5 μL of T4 ligase enzyme (200 U, NEB). The mix was incubated for 10 cycles of 5 min at 37 °C and 5 min at 23 °C. Five μL were transformed in 50 μL competent E. coli (Stbl3, Thermo fisher). Four colonies were picked and grown in LB + Carbenicillin, the plasmids purified (NEB Monarch Plasmid miniprep) and sequenced to verify correct insertion using U6_Fw_seq primer (Table 1 ).
Plasmids used in this study (from Addgene, USA)
| Plasmid construct | Plasmid name (Addgene) | Plasmid number (Addgene) |
|---|---|---|
| pLenti CMV:EGFP_PGK:Puro | pLenti CMV GFP Puro (658–5) | 17,448, [43 (link)] |
| pLenti hU6:gRNA_PKG:Puro | pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W | 67,974, [21 (link)] |
6
Cloning and Characterization of Wheat HSFA2h
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The full-length wheat HSFA2h sequence was amplified using the Fp_HSFA2h-pRI101 and Rp_HSFA2h-pRI101 primers (Table 1 ).
The amplified cDNA and pRI101-An vector (from Clontech) were restricted with NdeI and KpnI restriction enzymes, gel purified, and ligated with T4 ligase enzyme (NEB) in order to clone the HSFA2h into the plant expression vector pRI 101-AN under the regulation of the cauliflower mosaic virus (CaMV)-35S constitutive promoter. The recombinant plasmids were mobilized into Agrobacterium tumefaciens EHA105 and further used to transform A. thaliana ecotype Columbia by the floral dip method [39 ]. Antibiotic-resistant transformed lines were further validated through qRT-PCR and Northern blot analyses. Homozygous T3 lines were further characterized for their thermotolerance using different biochemical and molecular parameters under HS.
The amplified cDNA and pRI101-An vector (from Clontech) were restricted with NdeI and KpnI restriction enzymes, gel purified, and ligated with T4 ligase enzyme (NEB) in order to clone the HSFA2h into the plant expression vector pRI 101-AN under the regulation of the cauliflower mosaic virus (CaMV)-35S constitutive promoter. The recombinant plasmids were mobilized into Agrobacterium tumefaciens EHA105 and further used to transform A. thaliana ecotype Columbia by the floral dip method [39 ]. Antibiotic-resistant transformed lines were further validated through qRT-PCR and Northern blot analyses. Homozygous T3 lines were further characterized for their thermotolerance using different biochemical and molecular parameters under HS.
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