Megascript t7

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Megascript T7 is a laboratory instrument used for in vitro transcription. It is designed to synthesize large quantities of RNA from DNA templates containing the T7 promoter sequence.

Automatically generated - may contain errors

Lab products found in correlation

53 protocols using megascript t7

Preparation of qRT-PCR Copy Standards

Check if the same lab product or an alternative is used in the 5 most similar protocols

To obtain defined RNAs that serve as copy standards in qRT-PCR, partial 5'NTR sequences of NPHV and EPgV 1 were amplified and subsequently cloned into the plasmid pCR2.1 (Invitrogen). As template for PCR reaction, RNA obtained from the serum of a horse (designated "WST") with an EPgV 1/NPHV co-infection was used (Pfaender et al., 2014b) .
The cloned TDAV sequence was synthesized according to the only available TDAV sequence [KC145265] representing the amplicon generated with the previously published primers EVT-146/ EVT-146 (Chandriani et al., 2013) . BamHI linearized plasmids were used to transcribe run-off RNAs of defined size with a T7 transcription kit (MEGAscript T7, LifeTechnologies).
After removal of plasmid DNA by Turbo DNase (MEGAscript T7, LifeTechnologies) RNA was subsequently purified with the MegaClear kit (Ambion) and the amount of RNA was determined photometrically (NanoDrop). Mean values of three measurements were used to prepare RNA dilutions as copy standards for real-time RT-PCRs.

Postel A., Cavalleri J.M., Pfaender S., Walter S., Steinmann E., Fischer N., Feige K., Haas L, & Becher P. (2016). Frequent presence of hepaci and pegiviruses in commercial equine serum pools. Veterinary microbiology, 182.

+ Open protocol

+ Expand

Optimizing SARS-CoV-2 and GFP RNA Synthesis

Check if the same lab product or an alternative is used in the 5 most similar protocols

DNA oligos were purchased from Eurofins, and RNA oligos from IDT (Tables S1–5). The RNA donor was annealed to a DNA splint at 1:1 ratio in buffer (10 mM Tris-HCl pH 7.8, 50 mM NaCl). Annealing was performed at 95°C for 5 min and ramp down 0.1°C/sec to 25°C (Table S2). In vitro transcribed RNA of SARS-CoV-2 N-gene was synthesized with MEGAscript T7 (Thermo Fisher Scientific) using PCR product generated from SARS-CoV-2 WA1 cDNA and primers N0.5_F and N3-5_R (Eurofins) (Table S3). In vitro transcribed GFP RNA was synthesized with MEGAscript T7 (Thermo Fisher Scientific) using PCR product generated from pTE3’2J-GFP plasmid and primers T7_SINV_sgmRNA_F and SINV_polyA_R (Eurofins) (Table S3). Transcribed RNAs were purified using the Monarch RNA Cleanup kit (NEB).

Nemudryi A., Nemudraia A., Nichols J.E., Scherffius A.M., Zahl T, & Wiedenheft B. (2023). CRISPR-based engineering of RNA viruses. bioRxiv.

+ Open protocol

+ Expand

Synthesizing and Purifying SARS-CoV-2 and GFP RNAs

Check if the same lab product or an alternative is used in the 5 most similar protocols

DNA oligos were purchased from Eurofins, and RNA oligos were purchased from Integrated DNA Technologies (tables S1 to S5). The RNA donor was annealed to a DNA splint at a 1:1 ratio in buffer [10 mM tris-HCl (pH 7.8) and 50 mM NaCl]. Annealing was performed by incubating samples at 95°C for 5 min and then cooling down to 25°C at −0.1°C/s rate (table S2). In vitro transcribed RNA of SARS-CoV-2 N-gene was synthesized with MEGAscript T7 (Thermo Fisher Scientific) using PCR product generated from SARS-CoV-2 WA1 cDNA and primers N0.5_F and N3-5_R (Eurofins) (table S3). In vitro transcribed GFP RNA was synthesized with MEGAscript T7 (Thermo Fisher Scientific) using PCR product generated from pTE3′2 J-GFP plasmid and primers T7_SINV_sgmRNA_F and SINV_polyA_R (Eurofins) (table S3). Transcribed RNAs were purified using the Monarch RNA Cleanup Kit (NEB).

Nemudryi A., Nemudraia A., Nichols J.E., Scherffius A.M., Zahl T, & Wiedenheft B. (2023). CRISPR-based engineering of RNA viruses. Science Advances, 9(37), eadj8277.

+ Open protocol

+ Expand

Generating siRNA using Giardia Dicer

Check if the same lab product or an alternative is used in the 5 most similar protocols

siRNA was generated using baculovirus produced giardia Dicer as described³⁴. Briefly 1 ug of PCR product was in vitro transcribed using Megascript T7 (Ambion) and digested using dicer at 37C for 16 hours. siRNA was purified using Purelink RNA mini Kit (Ambion), absence of >22nt RNA was verified using gel electrophoresis and ethidium bromide staining. NCCIT cells were plated onto matrigel coated 24-well plates, transfected using 1.5 uL of RNAi-max (Invitrogen) in optimem (Gibco) with 25nM siRNA concentrations for 4 hours before addition of fresh media. siRNA knockdowns were performed for three consecutive days, cells were harvested 24 hours after final transfection. Two independent siRNA pools were generated for OCT4, NANOG, SOX2, one each for turboRFP (non-targeting control) and Rec, which overlaps the Env ORF. Primers used to generate dsRNA templates are listed in Supplementary Table #10.

Grow E.J., Flynn R.A., Chavez S.L., Bayless N.L., Wossidlo M., Wesche D., Martin L., Ware C., Blish C.A., Chang H.Y., Reijo Pera R.A, & Wysocka J. (2015). Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature, 522(7555), 221-225.

+ Open protocol

+ Expand

Generating siRNA using Giardia Dicer

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

CRISPR-Mediated Editing of dsx Exons

Check if the same lab product or an alternative is used in the 5 most similar protocols

Specific target sites on exons harboring alternative splicing of dsx were selected, specifically, sgRNA targeting exon 2 was designed to target the common region, exon 4 for the female-specific region, and exon 5 for the male-specific region. All the gRNAs were designed using an online tool, CRISPRdirect (http://crispr.dbcls.jp/, accessed on 20 November 2014) [48 (link)]. We designed gRNAs targeting Sfdsx^C, Sfdsx^F, and Sfdsx^M sites following the rule of 5′-GG-(N)18-NGG-3′ on dsx. The Sfdsx^C, Sfdsx^F, and Sfdsx^M gRNA sequences were designed with a length of 20 bp and aligned with S. frugiperda genome sequence (ASM1297921v2) to determine their specificity. The sgRNAs were sub-cloned and ligated to the pJET1.2 vector (ThermoFisher Scientific, Waltham, MA, USA). sgRNAs were synthesized using MEGAScript T7 (Ambion, Austin, TX, USA) in vitro following the manufacturer’s instructions. The TrueCut^TM Cas9 Protein (Invitrogen, Carlsbad, CA, USA) was purchased commercially and stored at −80 °C for experimental use.

Gu J., Wang J., Bi H., Li X., Merchant A., Zhang P., Zhang Q, & Zhou X. (2022). CRISPR/Cas9-Mediated Mutagenesis of Sex-Specific Doublesex Splicing Variants Leads to Sterility in Spodoptera frugiperda, a Global Invasive Pest. Cells, 11(22), 3557.

+ Open protocol

+ Expand

Luciferase-Based Hepatitis C Virus Replicon Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plasmids containing the sequence corresponding to subgenomic JFH-1 replicons bearing a luciferase reporter gene have previously been described [64 (link)]. After digestion with the restriction enzyme MluI, the linearized plasmids were in vitro transcribed using a commercial kit (Megascript T7; Ambion, Paisley, UK). The resulting products were digested with DNAse and precipitated with LiCl. Pelleted RNA was washed with 75% and 100% ethanol, and resuspended in nuclease-free water. In vitro transcribed RNA was transfected with Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) using the manufacturer´s recommendations. Luciferase activity was measured using a commercial kit (Dual Luciferase Assay System; Promega) at different times post-transfection. HCV polymerase inhibitor sofosbuvir (SFB; MedChem Express, Sollentuna, Sweden) was used as control [65 (link)].

Castro V., Calvo G., Ávila-Pérez G., Dreux M, & Gastaminza P. (2019). Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle. Cells, 8(11), 1456.

+ Open protocol

+ Expand

Drosophila S2 Cell Experiments

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

S2 cells, wild-type S2R+ cells, and tsc2 KO S2R+ cells [44 (link), 45 (link)] were grown in Schneider’s Drosophila Media (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and penicillin–streptomycin. For insulin treatment, 25 µg/ml of insulin was treated for the indicated time lengths. For rapamycin (LC Laboratories) treatment, cells were incubated with 20 nM rapamycin for the indicated time lengths.
For RNAi experiments, PCR templates for dsRNA against CCT1 through CCT8 were prepared using primers designed by SnapDragon-dsRNA design (http://www.flyrnai.org/snapdragon). dsRNAs for CCT1-8 were generated by PCR using MEGAscript T7 (Ambion) and purified using MEGAClear (Ambion). Thirty micrograms of dsRNA was treated in S2R+ cells in six-well plates for 3 days using bathing method [46 (link)].
For immunoprecipitation between Myc-CCT4 and Rheb-V5 proteins, coding sequences of CCT4 and Rheb were cloned into pAc5.1-V5/His (Invitrogen) with N-terminal Myc tag with and without C-terminal stop codon, respectively. The cloned constructs were transfected in S2 cells using Effectene reagent (Qiagen).

Kim A.R, & Choi K.W. (2019). TRiC/CCT chaperonins are essential for organ growth by interacting with insulin/TOR signaling in Drosophila. Oncogene, 38(24), 4739-4754.

+ Open protocol

+ Expand

Quantitative RT-PCR Analysis of Viral and Host Transcripts

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was extracted from different tissues (0.1 g liver or tissues indicated in Supplementary information, Figure S1D), cells (1 × 10⁶) or serum (0.1 ml) using Trizol or Trizol LS reagent (Invitrogen). Quantitative RT-PCR analyses with One-step QuantiTect SYBR Green Kit (Qiagen, CA, USA) of the indicated genes were performed as previously described^{14 (link)} in an ABI 7500 (ABI, CA, USA).The internal standard HCV RNA was synthesized by in vitro transcription (MEGAscript T7, Ambion, TX, USA) from pJFH-1 plasmid and quantified by a commercial kit (path-HCV, PrimerDesign, UK) at 1.1 × 10¹⁰ copies/ml. Serial dilutions of the internal standard HCV RNAs were spiked in ICR sera and liver homogenates to determine the linear range of detection and the limit of detection, using One-step QuantiTect SYBR Green Kit. The limit of detection of HCV RNA by this method is defined as the lowest concentration at which 95% of HCV RNA are detected^{53 (link)}, i.e., 500 copies/ml (serum) and 100 copies/mg (liver), respectively. Nested PCR was performed to detect HCV negative strand^{16 (link)} (see Supplementary information, Table S6 for primers). Quantitative RT-PCR analyses were carried out similarly for mRNA levels of IFNα4, IFNβ1, ISGs, miR-122, apoE, CypA and IL-28 (see Supplementary information, Table S6 for primers). Data were analyzed with ABI software version 2.0.3 (Applied Biosystems).

Chen J., Zhao Y., Zhang C., Chen H., Feng J., Chi X., Pan Y., Du J., Guo M., Cao H., Chen H., Wang Z., Pei R., Wang Q., Pan L., Niu J., Chen X, & Tang H. (2014). Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice. Cell Research, 24(9), 1050-1066.

+ Open protocol

+ Expand

Transfection of Hepatitis C Replicon RNA

Check if the same lab product or an alternative is used in the 5 most similar protocols

A plasmid containing the sequence corresponding to a subgenomic JFH-1 replicon bearing a firefly luciferase reporter gene was kindly provided by Dr. Ralf Bartenschlager (U. of Heidelberg) [52 (link)]. After digestion with the restriction enzyme MluI, the linearized plasmid was transcribed in vitro using a commercial kit (Megascript T7; Ambion-Paisley, UK). The resulting products were digested with DNAse and precipitated with LiCl. Pelleted RNA was washed with 75% and 100% ethanol, and resuspended in nuclease-free water. In vitro transcribed RNA was transfected into the different cell lines together with a plasmid expressing Renilla luciferase under a minimal promoter (pRL-null; Clontech-California, USA) using Lipofectamine 2000 and the manufacturer´s recommendations (Life Technologies- California, USA). Firefly and Renilla luciferase activities were measured in the sample using a commercial kit (Dual Luciferase Assay System; Promega-Wisconsin, USA) at different times post-transfection.

Mingorance L., Castro V., Ávila-Pérez G., Calvo G., Rodriguez M.J., Carrascosa J.L., Pérez-del-Pulgar S., Forns X, & Gastaminza P. (2018). Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation. PLoS Pathogens, 14(9), e1007284.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!