Template cDNAs (wildtype or mutant human Kv7.1 in pSGEM, wildtype human KCNE1 in pSP64) were linearized with a suitable restriction enzyme (Nhe I/pSGEM, Eco RI/pSP64). cRNA was synthesized from 1 μg linearized cDNA by in vitro transcription (mMessage mMachine T7 or SP6 kit, Ambion, Life Technologies Darmstadt, Germany). cRNA concentrations were determined by photometry (Nanodrop, Peqlab, Erlangen, Germany) and cRNA quality was checked by agarose gel electrophoresis.
Mmessage mmachine t7 or sp6 kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The MMessage mMachine T7 or SP6 kit is a laboratory tool used for in vitro transcription. The kit provides the necessary components, including enzymes and buffers, to synthesize capped and polyadenylated mRNA from DNA templates containing the appropriate promoter sequences.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using mmessage mmachine t7 or sp6 kit
1
Synthesis of Kv7.1 and KCNE1 cRNAs
2
Capped Sense RNA Injection Protocol
3
Generating cRNA of Mklp1, Eb3-eGFP, and Gap43-eGFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full-length clone of mouse Mklp1 (MGC: 54492) was purchased from Open Biosystems and amplified for insertion into pIVT-eGFP or pIVT-mCherry 60 (link). Gap43-eGFP was a gift from Dr. Greg FitzHarris (U. of Montreal). To generate cRNA of Eb3-egfp61 (link), Mklp1-eGfp, Mklp1-mCherry, and Gap43-eGfp, plasmids were linearized and transcribed in vitro using mMessage mMachine T7 or SP6 kit (Ambion, Cat# AM1344) according to manufacturer’s protocol.
cRNA was purified using SeraMag Speedbead (Sigma Aldrich, Cat# GE65152105050250) nucleotide purification method previously described 62 (link). Briefly, in vitro transcription reaction solution was brought up to 150 μl and mixed with 100 μl of magnetic beads and let stand for 5 minutes. Beads were then pelleted using a magnetic stand and washed with 80% ethanol. cRNA was eluted using 20 μl nuclease-free water and stored at −80° C.
cRNA was purified using SeraMag Speedbead (Sigma Aldrich, Cat# GE65152105050250) nucleotide purification method previously described 62 (link). Briefly, in vitro transcription reaction solution was brought up to 150 μl and mixed with 100 μl of magnetic beads and let stand for 5 minutes. Beads were then pelleted using a magnetic stand and washed with 80% ethanol. cRNA was eluted using 20 μl nuclease-free water and stored at −80° C.
4
Cloning and mRNA Rescue Experiment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length coding sequence DNA of trip11, dnhd1, cfap74, and egr4 were synthesized by the company GENEWIZ (Suzhou, China) and then subcloned into the pCS2+ vector. The positive clones were selected by DNA sequencing to be applied for generating full-length mRNAs. The corresponding mRNAs of candidate genes were generated by T7 or SP6 mMessage mMachine kit (Ambion, America). For the rescue experiment, the mRNA and MO of candidate genes were mixed and injected into one-cell-stage embryos. The dose of mRNAs and MOs are listed in S11 Table .
5
Zebrafish Embryonic Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zebrafish total RNA was extracted from 1 to 2 days post fertilization (dpf) wild-type embryos (AB strain) and then was retrotranscribed into coding DNA (cDNA) with PrimeScripttm RT reagent (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. The full-length coding sequence DNA of tctn2, pacrg, galnt11, numb, and rnf115 was amplified using specific primers with restriction enzyme digestion loci, which were next subcloned into the pCS2+ vector (Additional file 1 : Table S2). Positive clones selected by DNA sequencing were applied to generate the corresponding full-length mRNAs using a T7 or SP6 mMessage mMachine kit (Ambion). For the over-expression experiment, pacrg, tctn2, numb, and galnt11 mRNAs were injected into one-cell-stage embryos and green fluorescent protein (GFP) was used as a negative control; for the rescue experiment, 6.25 pg rnf115 mRNA was injected into one-cell-stage embryos mixed with 2 pg rnf115 MO.
6
Cloning and Expression of GFP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dresden University of Technology, Germany), pCS2-GFP-rGBD (a kind gift from William Bement), pSp64T-C-cad Ctail [16] (link), pT7T-ZO-1-GFP [17] (link) and pT7T-SF9-GFP [18] (link) were used in this study. pT7T-GFP-Rock II K/R was constructed by site-directed mutagenesis of lysine 117 (K117) from Xenopus Rock II (a kind gift from Johné Liu, [19] (link)). The K117, essential for the catalytic activity of the kinase, was replaced by an arginine to abolish Rock II activity because the overexpression of the active kinase causes division defects, which are not obtained with the Rock II K/R mutant. The amplification product was cloned into the vector pT7T in phase with N-terminal GFP.
PT7T-3xGFP-anillin was constructed by PCR amplification of the Xenopus anillin sequence, from the plasmid pT7T-GFP-Xlanillin [15] (link) and subcloned into pT7T-3xGFP with N-terminal 3xGFP. All new constructs were verified by sequencing. All plasmids were linearised to generate mRNAs by in vitro transcription using the T7 or SP6 mMessage mMachine kit (Ambion).
PT7T-3xGFP-anillin was constructed by PCR amplification of the Xenopus anillin sequence, from the plasmid pT7T-GFP-Xlanillin [15] (link) and subcloned into pT7T-3xGFP with N-terminal 3xGFP. All new constructs were verified by sequencing. All plasmids were linearised to generate mRNAs by in vitro transcription using the T7 or SP6 mMessage mMachine kit (Ambion).
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!