MOs (5′-CCGCCCTGGCAAGTGCGGACGA-3′) for depleting murine Cyclin B2 were synthesized by Gene Tools according to mCCNB2 (NM_007630 ; encoding Ccnb2; Gui and Homer, 2013 (link)). H2B-mCherry and MAP7-EGFP cRNAs were made from a pMDL-H2B-mCherry vector and a pGEM-MAP7-EGFP vector through in vitro transcription (T3 or T7 mMessage mMachine [Ambion] according to the manufacturer’s instructions), respectively. Mouse Ccnb2 gene (NM_007630 .2) was cloned into a pcDNA3.1-Venus vector, and its cRNA was prepared using T7 mMessage mMachine (Ambion). Mouse Ccnb1 gene (NM_172301 .3) was cloned into a pCS2+ vector, and its cRNA was prepared using SP6 mMessage mMachine (Ambion). All cRNAs were purified with RNeasy Mini kits (QIAGEN), dissolved in nuclease-free water, and stored at −80°C. A concentration of 500 ng/µl was used for microinjection. Microinjection was performed with a Nikon operating system.
T7 mmessage mmachine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T7 mMessage mMachine is a laboratory instrument used for in vitro transcription of RNA. It is designed to generate capped and polyadenylated mRNA from linearized DNA templates with high efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Sp6 transcription kit, by Thermo Fisher Scientific (4 mentions)
Quikchange 2, by Qiagen (2 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Xbai, by New England Biolabs (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pcdna5 to vector, by Thermo Fisher Scientific (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Phenol red, by Merck Group (1 mentions)
Oc 725c oocyte clamp, by Warner Instruments (1 mentions)
Prism 5, by GraphPad (1 mentions)
Nucleofector kit t, by Lonza (1 mentions)
Amaxa nucleofector, by Lonza (1 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Microspin g 25 column, by GE Healthcare (1 mentions)
Transmessenger transfection reagent, by Qiagen (1 mentions)
Megashortscript t7, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
30 protocols using t7 mmessage mmachine
1
Murine Cyclin B2 Depletion Protocol
2
Overexpression of KDM2A in Zebrafish
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Zebrafish embryos of the TLF (Tüpfel long fin) strain were injected at the one cell stage with 3.6 nl of capped polyadenylated mRNA (mMESSAGE mMACHINE T7, Ambion) generated from human FLAG-GFP-tagged KDM2A constructs (see Supplementary Methods ) at a concentration of 62.5 ng/μl combined with 0.05% Phenol Red (Sigma). Embryos were raised at 28.5°C for 24 h and then characterized based on their morphology. Dead embryos and embryos with abnormal phenotypes were classified into the abnormal category.
3
Electrophysiological Analysis of Xenopus Odorant Receptors
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Xenopus oocyte collection and preparation and electrophysiological recordings were conducted following previously reported protocols [60 (link), 61 (link)]. The PxylOR16, PxylOR27, and PxylOrco cRNAs were synthesized using mMESSAGE mMACHINE T7 (Ambion, Austin, TX). PxylOR16 or PxylOR27 cRNAs (27.6 ng each) were simultaneously microinjected with PxylOrco cRNA into mature healthy Xenopus oocytes (stages V–VII). After injection, oocytes were incubated for 4–7 days at 16 ℃ in 1 × Ringer’s solution supplemented with 5% dialyzed horse serum, 50 μg/mL tetracycline, 100 μg/mL streptomycin, and 550 μg/mL sodium pyruvate. Odorant-induced currents were recorded with an OC-725C oocyte clamp (Warner Instruments, Hamden, CT) at a holding potential of − 80 mV. Oocytes were exposed to compounds in ascending order of concentration with an interval between exposures that allowed the current to return to baseline. The data were acquired and analyzed with Digidata 1440A and pCLAMP 10.2 software (Axon Instruments Inc., Union City, CA). Statistical comparison of responses of oocytes to tested ligands and dose–response data were analyzed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA).
4
Synthesizing and Injecting Rspo3 mRNA
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Capped mRNA of different target genes was synthesized with mMESSAGE mMACHINE@T7 (Ambion, Foster City, CA, USA). The primers were CS-Rspo3-mRNA-Fw and CS-Rspo3-mRNA-Rv (Table 1 ). Microinjection was carried out on Harvard Apparatus PLI-100 (NatureGene, NV, USA) machine in one- to four-cell-stage embryos with 1 nl Rspo3 mRNA for each embryo. Embryos not injected were the control group.
5
Engineering SARS-CoV Mutants via Recombineering
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Using “en passant recombineering” (recombineering by
mutagenesis) (54 (link)), mutations in
the nsp10-, nsp14-, and nsp16-coding regions of SARS-CoV isolate
Frankfurt-1 were engineered in prSCV, a pBeloBac11 derivative containing
a full-length cDNA copy of the viral genome (55 (link)). The DNA of such BAC clones was
linearized with NotI, extracted with phenol-chloroform, and transcribed
with the mMessage-mMachine® T7 (Ambion) using 2 μg of
DNA template in a 20-μl reaction. Full-length viral RNA was
precipitated with LiCl according to the manufacturer's protocol, and 6
μg was electroporated into 5 × 106BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after
>4 h of induction with 2 μm doxycycline (53 (link)). Electroporation was done using
the Amaxa Nucleofector (Lonza), Nucleofector Kit T, and program T-020
according to the manufacturer's instructions. Cells were mixed in a 1:1
ratio with Vero-E6 cells and seeded on coverslips for immunofluorescence
microscopy and for analysis of virus production. Each mutant was
launched twice from independently generated BAC clones. All work with
live SARS-CoV was performed inside biosafety cabinets in a biosafety
level 3 facility at Leiden University Medical Center.
mutagenesis) (54 (link)), mutations in
the nsp10-, nsp14-, and nsp16-coding regions of SARS-CoV isolate
Frankfurt-1 were engineered in prSCV, a pBeloBac11 derivative containing
a full-length cDNA copy of the viral genome (55 (link)). The DNA of such BAC clones was
linearized with NotI, extracted with phenol-chloroform, and transcribed
with the mMessage-mMachine® T7 (Ambion) using 2 μg of
DNA template in a 20-μl reaction. Full-length viral RNA was
precipitated with LiCl according to the manufacturer's protocol, and 6
μg was electroporated into 5 × 106BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after
>4 h of induction with 2 μ
the Amaxa Nucleofector (Lonza), Nucleofector Kit T, and program T-020
according to the manufacturer's instructions. Cells were mixed in a 1:1
ratio with Vero-E6 cells and seeded on coverslips for immunofluorescence
microscopy and for analysis of virus production. Each mutant was
launched twice from independently generated BAC clones. All work with
live SARS-CoV was performed inside biosafety cabinets in a biosafety
level 3 facility at Leiden University Medical Center.
6
Microinjection of Bdkrb2 mRNA in Zebrafish
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Capped mRNAs of two Bdkrb2 genes (Ssc_10023113 and Ssc_10023117) were synthesized with mMESSAGE mMACHINE@T7 (Ambion, Foster City, CA, USA). The primers were Ssc_10023113_mRNA_Fw/ Rv and Ssc_10023117_mRNA_Fw/Rv (Additional file 9 : Table S3). Microinjection was performed on arvard Apparatus PLI-100 (NatureGene, NV, USA) machine in one- to four-cell-stage embryos of transgenic strain (Fli1a: EGFP) with 1.5 ng Bdkrb2 mRNA of black rockfish for each embryo. Embryos without injection and with injection of sterilized DEPC water were the control groups.
7
Rat GABAAR Subunit Expression
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DNA for wild-type GABAAR rat α1, β2, and γ2L subunits were a gift from Dr. Cynthia Czajkowski. Single alanine substitutions were introduced throughout the α1M2–M3 linker from L276-T283 in addition to the gain of function α1L9'T pore mutation (rat α1L263T) (QuikChange II, Qiagen). Mutations V279D and V279W were introduced similarly. Each construct was verified by forward and reverse sequencing of the entire gene. mRNA for each construct was generated (mMessage mMachine T7, Ambion) for expression in X. laevis oocytes (EcoCyte Bioscience, Austin, TX). Oocytes were injected with 27–54 ng of total mRNA for α, β, and γ subunits in a 1:1:10 ratio (Boileau et al., 2002 (link)) (Nanoject, Drummond Scientific). Oocytes were incubated in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, pH 7.2) with 100 mg/ml gentamicin at 18°C.
8
Expressed GABA Receptor Variants
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DNA for wild-type GABAAR rat a1, b2 and g2L subunits were a gift from Dr. Cynthia Czajkowski. Single alanine substitutions were introduced throughout the a1M2-M3 linker from L276-T283 in addition to the gain of function a1L9'T pore mutation (rat a1L263T) (QuikChange II, Qiagen). Each construct was verified by forward and reverse sequencing of the entire gene.
mRNA for each construct was generated (mMessage mMachine T7, Ambion) for expression in Xenopus laevis oocytes (EcoCyte Bioscience, Austin, TX). Oocytes were injected with 27-54 ng of total mRNA for a, b and g subunits in a 1:1:10 ratio (Boileau et al., 2002) (Nanoject, Drummond Scientific). Oocytes were incubated in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5
HEPES, pH 7.2) with 100 mg/ml gentamicin at 18°C.
mRNA for each construct was generated (mMessage mMachine T7, Ambion) for expression in Xenopus laevis oocytes (EcoCyte Bioscience, Austin, TX). Oocytes were injected with 27-54 ng of total mRNA for a, b and g subunits in a 1:1:10 ratio (Boileau et al., 2002) (Nanoject, Drummond Scientific). Oocytes were incubated in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5
HEPES, pH 7.2) with 100 mg/ml gentamicin at 18°C.
9
In Vitro Transcription and Transfection of mRNA
10
Oocyte Expression of HCN4 Channels
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For oocyte expression, the previously described cDNA template hHCN4-WT/pSGEM was linearized by NheI [58 (link)], and cRNA was generated using the in vitro transcription kit mMessage mMachine T7 (Life Technologies, Darmstadt, Germany). Defolliculated oocytes stage V and VI were purchased from EcoCyte Bioscience (Dortmund, Germany) and injected, each with 50.6 nL containing 5 ng of HCN4 cRNA. After injection, oocytes were stored for 4–5 days at 18 °C in Barth solution containing the following (mmol/L): NaCl, 88; KCl, 1; CaCl2, 0.4; Ca(NO3)2, 0.33; MgSO4, 0.6; TRIS-HCl, 5; NaHCO3, 2.4 and (mg/L) theophylline, 80; benzylpenicillin, 63; streptomycin, 40; gentamycin, 100.
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