Expression plasmids containing a His6 tag at the C terminus were constructed as follows. Viral RNA was isolated from DENV-2 (S16803) infected Vero cell supernatant. Primers used for the NS3 protease (NS3Pro) fragment were: forward 5’-CATATG GAAGAACA-AACACTGACCATACTC-3’ and reverse 5’-GCGGCCGC ATGGAGGTCCATGATG-gtcagtc-3’ containing Nde I and Not I restriction sites, respectively. These primers generated a 708 bp fragment containing the last 40 aa of NS2b as well as amino acids 1–196 of NS3 (protease domain). Primers for the NS3 helicase (NS3Hel) fragment were: forward 5’-CATATG GACAACCCAG-AGATCGAAGATGAC-3’ and reverse 5’-GCGGCCGC- GTCTGCGTAGTTGATGCCTTCAGC-3’ and generated a 1140 bp fragment containing aa 174 to aa 554 of the helicase domain. The RT-PCR reactions were performed using the SuperScript III Platinum One-Step RT-PCR kit (Invitrogen) and the products were cloned into the TOPO-TA vector (Invitrogen). The positive clones were identified, and the plasmid DNAs were digested with Nde I and Not I. The DENV-2 NS3pro and NS3hel fragments were then subcloned into the expression vector pET-30a (+) (Novagen) at the Nde I and Not I sites upstream of the C-terminal 6 x His tag. In this vector the inserts are under the control of the lac promoter, which is inducible with IPTG. The integrity of both constructs was verified by sequencing.
Superscript 3 platinum one step rt pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperScript III Platinum One-Step RT-PCR kit is a reagent system designed for the reverse transcription and amplification of RNA targets in a single reaction. It includes the necessary components for both the reverse transcription and PCR steps.
Automatically generated - may contain errors
Lab products found in correlation
Topo ta vector, by Thermo Fisher Scientific (1 mentions)
Nucleospin dx virus kit, by Macherey-Nagel (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Magna pure compact, by Roche (1 mentions)
Viral na small volume kit, by Roche (1 mentions)
Lightcycler 480 thermocycler, by Roche (1 mentions)
Magna pure 96, by Roche (1 mentions)
5 protocols using superscript 3 platinum one step rt pcr kit
1
Cloning DENV-2 NS3 Protease and Helicase
2
Quantitative RT-PCR for Luciferase Encapsidated RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Luciferase encapsidated RNA was extracted using Nucleospin Dx Virus kit (Macherey-Nagel) from 150 µL of pseudotype suspension, according to the manufacturer's instructions. Quantitative RT-PCR targeting the luciferase gene was carried out using the following primers [forward 5′-ACACCCCAACATCTTCGAC-3′ , Reverse 5′-TCGCGGTTGTTACTTGACTG-3′] and probe [5′-FAM-TTGGAGCACGGAAAGACGATGAC-BHQ1-3′] (BHQ1: black hole quencher 1) with a LightCycler 480 instrument (Roche) and a SuperScript III Platinum OneStep RT-PCR kit (Invitrogen). The reactions were incubated in a 96-well optical plate at 45°C for 15 min, then 95°C for 3 min, followed by 50 cycles of 95°C for 10 sec, 50°C for 10 sec and 72°C for 20 sec and a cooling step at 40°C for 30 sec.
3
SARS-CoV-2 E-gene RT-PCR Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the HCB, all samples were inactivated with 1:1 volume of Cobas Omni Lys (Roche, Germany), and total nucleic acid was extracted using MagNA Pure Compact (Roche, Switzerland). Respiratory samples and elutes were aliquoted and stored at −80°C.
Extracted RNAs were tested for the presence of Envelope (E) sgRNA using the leader-specific primer described by Wölfel et al. (9 (link)) as well as primers and probes targeting sequences downstream of the start codons of the E gene (13 ) (see Text S2 and Table S1 athttps://doi.org/10.6084/m9.figshare.16802200.v1 ). RT-PCR was performed using the SuperScript III Platinum One-Step RT-PCR kit (Invitrogen) with a primer concentration of 400 nM and a probe concentration of 200 nM. The CT cutoff for negative samples was >40.
Extracted RNAs were tested for the presence of Envelope (E) sgRNA using the leader-specific primer described by Wölfel et al. (9 (link)) as well as primers and probes targeting sequences downstream of the start codons of the E gene (13 ) (see Text S2 and Table S1 at
4
Multiplex RT-PCR for Arboviral Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We extracted RNA from whole blood, serum, urine, and semen fractions with the MagNA Pure 96 instrument using the DNA and viral NA small volume kit (Roche Diagnostics) with 200 μL input and 100 μL output minimum volumes. For semen cell fractions, we adjusted input volume to 2 × 106 cells. We detected DENV RNA using a homemade 1-step real-time RT-PCR triplex protocol to specifically detect DENV, ZIKV, or chikungunya (CHIKV) RNA (18 (link)). We added 15 μL of extracted RNA to a 45 μL amplification containing 140 nmol/L of each primer; 100 nmol/L each of GAPDH-LC670, DENV-LC610, and ZIKV-FAM TaqMan probes; 45 nmol/L of CHIKV-Cyan500 TaqMan probe; and 1 μL of enzyme (Superscript III Platinum One-step RT-PCR kit, ThermoFisher Scientific, https://www.thermofisher.com ). We used a LightCycler 480 Thermocycler (Roche Diagnostics) for amplification and detection with reverse transcription at 52°C for 20 min, melting at 95°C for 2 min, followed by 45 cycles with denaturation at 95°C for 15 s, hybridization at 55°C for 45 s, elongation at 68°C for 20 s, and finally, cooling at 40°C for 30 s.
5
Detection of Infectious Bronchitis Virus in Pooled Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 7 pooled swab samples and the 5 pooled tracheal tissues were analyzed by RT-PCR. RNA extraction from the samples was done using QIAampi® RNA micro kit [Qiagen Ltd., Hilden, Germany] as per the manufacturer’s instructions. The pools were tested for IBV presence using the SuperScript™ III Platinum™ One-Step RT-PCR Kit (Thermo Fisher, Waltham, MA, USA), amplifying a 464 bp hyper-variable region of the S1 gene using the method described by Adzhar et al,22 (link) while a method described by Jahantigh et al23 was used for Nested PCR (Table 1 ).
Primers to Identify IBV Serotypes in Reverse Transcription Nested PCR
| PCR types | Primer Designation | Sequence | Serotype | Fragment size (bp) | Reference |
|---|---|---|---|---|---|
| Reverse transcription PCR | XCE2+ | 5’-CACTGGTAATTTTTCAGATGG-3’ | Universal | 466 bp | [22 (link)] |
| XCE2- | 5’-CCTCTATAAACACCCTTGCA-3’ | ||||
| Reverse transcription Nested PCR | XCE3- | 5’-CAGATTGCTTACAACCACC-3’ | – | Universal | |
| DCE1+ | 5’-TTCCAATTATATCAAACCAGC-3’ | D274 | 217 bp | [32 (link)] | |
| MCE1+ | 5’-AATACTACTTTTACGTTACAC-3’ | Mass | 295 bp | ||
| BCE1+ | 5-AGTAGTTTTGTGTATAAACCA-3’ | 4/91 | 154 bp |
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!