cDNAs representing the entire MHV-A59 genome, carrying either the wild-type sequence or mutations in SL5C or SL6, were constructed by sequential ligation of digested and gel purified cDNAs A to G and the ligated cDNA transcribed in vitro using the Ambion T7 mMESSAGE mMACHINE kit (Applied Biosystems), as described previously (Johnson et al., 2005 (link), Yount et al., 2002 (link)). The transcription reactions were then electroporated into BHK-R cells that were then overlaid onto DBT cells. Cultures were incubated for up to 72 h and monitored by phase microscopy for the development of cytopathic effect (CPE). Recovered viruses were plaque purified on L2 cell monolayers and subsequently propagated in DBT cells. The mutated region and the flanking region (nts 20–500) were amplified by RT-PCR and sequenced to confirm the presence of the introduced mutations and to identify any second-site mutations in the neighborhood that might have arisen during virus recovery and propagation. For each mutant, at least two (usually 3) plaque-purified isolates at passage 1 (P1) were selected for sequencing.
Ambion t7 mmessage mmachine kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion T7 mMESSAGE mMACHINE kit is a lab equipment product used for in vitro synthesis of capped and polyadenylated mRNA transcripts. The kit includes T7 RNA polymerase and other necessary reagents to generate mRNA from DNA templates.
Automatically generated - may contain errors
4 protocols using ambion t7 mmessage mmachine kit
1
Murine Coronavirus Genome Manipulation
2
Reverse Genetics of Murine Coronavirus MHV-A59
3
Functional Expression of Kv4.3 and KChIP
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Amino-terminal Myc-tagged KV4.3 (Myc-KV4.3) was generated by subcloning human KV4.3 cDNA into the pcDNA3.1-Myc vector (Invitrogen, Carlsbad, CA, USA). The KV4.3 p.R419H variant was created by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Amino-terminal HA-tagged KChIP2 and KChIP3 (HA-KChIP2 and HA-KChIP3) were created by subcloning human KChIP2 and KChIP3 cDNAs, respectively, into the pcDNA3-HA vector (Invitrogen, Carlsbad, CA, USA). All the constructs were verified by DNA sequencing. For in vitro transcription, appropriate restriction enzymes were applied to linearize cDNAs, from which capped cRNAs were transcribed using the Ambion mMessage mMachine T7 kit (Thermo Scientific, Waltham, MA, USA).
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Oocyte Injection of TRPV1 Constructs
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DNA constructs (wt TRPV1, eTRPV1, e1TRPV1, and e2TRPV1) and single-cysteine mutants in pMO were linearized using PmeI. mRNAs were prepared from linearized DNAs using the Ambion mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific) and purified using the RNeasy mini kit (Qiagen) for oocytes injection.
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