TTT mRNA prep Kit – 555
ALL-IN-ONE Kit for Transcription, Transfection and Tracking
- Catalog No.
- 1 Kit
BCK-TTT mRNA 555
- 1100 €
Our TTT mRNA prep Kit, includes all reagents for transcription, dye labeling, transfection and tracking of your mRNA. Instead of buying several expensive kits for all these applications an ONE-IN-ALL kit was designed with an easy to handle protocol. This kit has been developed for the production of labeled mRNA by in vitro transcription (IVT) from bacteriophage T7 RNA polymerase promoter templates. The kit utilizes a proprietary nucleotide mixture to label the mRNA with alkynes. After transcription these alkynes can be reacted with azido-derivates of fluorescent dyes, haptenes and other labels in a highly selective fashion under benign click reaction conditions. Fluorescently labeled mRNA can be used to detect localization of the mRNA inside of cells after transfection. Furthermore, in order to control mRNA production, click labeling, transfection and protein expression, the kit provides a control DNA template that codes for an eGFP mRNA.
S.Croce, S. Serdjukow, T. Carell, T. Frischmuth, ChemBioChem, 2020, 21, 1-7
How does the DNA template influence in vitro transcription performance?
DNA template quality directly influences yield and quality of transcription reactions. Linearized plasmid DNA needs to be fully digested and should be free of contaminating RNase, protein and salts. We recommend selecting restriction enzymes that generate blunt ends or 5´-overhangs and purification after the enzymatic restriction (e.g. via silica-membrane based purification columns).
Alternatively, a PCR mixture can be used directly, but better yields will usually be obtained with purified PCR products (e.g. via silica-membrane based purification columns).
What do I need to consider when planning my transcription experiments using your kits?
Promotor choice: Different RNA polymerase promoters such as T7, T3 and SP6, are commonly used for the in vitro production of RNA. Our kits, TTT RNA prep and RNA labelling kit, are based on a T7 RNA polymerase promoter and will not function upon usage of a different one.
mRNA production: For the production of functional mRNA please ensure that the DNA template encodes the required structural features e.g. 3’-UTR, 5’-UTR, correctly orientated target sequence and ideally a poly A-tail. Alternatively, polyA-tailing can be introduced post-transcriptionally using a Poly(A) polymerase.
What is the promoter sequence of T7 RNA Polymerase?
5′ TAATACGACTCACTATAG 3′
T7 RNA polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Can the TTT mRNA Prep kit be used for other RNA?
No, this kit has been designed for preparation and labeling of mRNA from DNA templates, only. Due to the nucleotide mix present in the kit, the resulting mRNA will contain a CAP structure and pseudo-uridine for enhanced translation efficiency and intracellular stability, besides the clickable modification.
What kind of CAP is introduced with the TTT mRNA prep kit?
The CAP structure is introduced co-transcriptionally and results in a CAP 1 structure.
How much RNA can I expect after in vitro transcription using the TTT mRNA prep Kit?
Starting from 1 µg of DNA template, it is common to end up with a solution containing from 10 to 12 µg of mRNA.
What is the best way to handle RNA to avoid degradation?
Due to its chemical structure RNA is less stable than DNA. At high temperatures, under basic conditions (pH>9) and in the presence of high Mg2+ concentrations, chemical reactions that favour hydrolysis of the RNA molecule take place. Additionally, RNases are common contaminations in standard laboratory conditions compared to DNases. We recommend to clean the benches and equipment with RNase inhibitors or specific detergent mixtures and to wear fresh gloves when handling the tubes (gloves that have been worn or that e.g. touched door/refrigerator handles are considered contaminated). Moreover, RNase-free H2O and tubes should be used for buffer preparation and to dissolve the samples.
What is the best way to store RNA to avoid degradation?
For storage we recommend to store RNA at –80 °C, which should conserve RNA for one year without significant degradation. Alternatively.
In our experience, short synthetic RNAs (up to 50mer) are more stable than long single-stranded RNAs from biological samples and therefore can be stored at –20 °C in a dry format.
What kits are available from baseclick for RNA modification?
We offer several kits to prepare labelled RNAs that are based on enzymatic incorporation of alkyne- or azide-modified nucleotides and subsequent click labelling.
The RNA Labeling Kit allows you to introduce several alkyne groups randomly in your RNA through the incorporation of the 5-ethynyl-UTP (EUTP) by the T7 RNA polymerase during in vitro transcription.
The TTT mRNA prep Kit was developed and specifically designed for the production of alkyne decorated messenger RNA by the help of T7 RNA polymerase and EUTP.
The 3’END Labeling Kit introduces site specifically at the 3’ end of an RNA a single azide group via the incorporation azide-ATP derivative.
- How does the DNA template influence in vitro transcription performance?
12 months unopened after receipt
diverse, see kit boxes
kit system made of different components
please see user manual of the kit
- Shelf Life