LZCap™AG(3'Ma-Cy7) is a Cap1 analog with a Cy7 fluorescent marker, which can be used as an mRNA co-transcription capping reagent. Under the action of T7 polymerase, LZCap®AG(3'Ma-Cy7), NTPs and template DNA are co-transcribed to produce mRNA with a 5'-end Cap 1 structure. The capped mRNA can be directly translated and expressed in cells and in vivo, with excellent expression efficiency. The mRNA capped by LZCap®AG(3'Ma-Cy7) can be detected by flow cytometry and fluorescence microscopy for Cy7 fluorescence, and the distribution of mRNA and LNP can be traced and located. The recommended wavelength of Cy7 fluorescence of LZCap®AG(3'Ma-Cy7) mRNA is: (651/780). Specifications Cat.No. 10689ES10/10689ES60 Size 50 μL、100 μL Molecular Formula C68H86N18O30P4S2（Free acid） Molecular Weight 1823.55（Free acid） Concentration 25 mM Purity HPLC ≥90% Salt type NH4+ Structure Components Name 10689ES10 10689ES60 LZCap™AG(3'Ma-Cy7) 50 μL 100 μL Storage This product should be stored at -25~-15℃ for 2 years Instructions DNA template design of LZCap™ LZCap™AG(3'Ma-Cy7) is suitable for sequences starting with AG. The T7 promoter (underlined) followed by the AG sequence can effectively initiate transcription. Operating Instructions 1.Thaw the components needed for the experiment on ice. 2. Prepare the transcription system at room temperature according to the following reaction system. Component Volume(μL) Final concentration RNase Free Water Make up to 20 μL / ATP(100mM) 1 5mM UTP(100mM) 1 5mM CTP(100mM) 1 5mM GTP(100mM) 1 5mM LZCap®AG(3'Ma-Cy7) (25mM) 3.2 4mM 10×Transcription Buffer 2 1× Linear DNA 1μg 50ng/μL Recombinant RNase Inhibitor(40U/μL) 0.5 1U/μL Pyrophosphatase(0.1U/μL) 0.4 0.002U/μL T7 RNA polymers(250U/μL) 0.64 8U/μL Final volume 20μL 【Note】: *Modified N-Me-pUTP can be used in place of wild-type UTP.The modified N-Me-pUTP reduces theimmunogenicity of mRNA. 3. Mix the prepared reaction solution, centrifuge briefly, and incubate at 37℃ for 2-3 h. If the transcript length is less than 100 nt,increase the reaction time to 4-8 h. 3. Mix the prepared reaction solution, centrifuge briefly, and incubate at 37℃ for 2-3 hours. If the transcript length is less than 100nt, increase the reaction time to 4-8 h. Notes: 1) LZCap™AG (3'Ma-Cy7) is suitable for T7 promoter transcription vectors with 5'AG 3' start sequence, which needs to be considered when constructing the vector. 2) LZCap™AG (3'Ma-Cy7) and its mRNA products should be stored and used away from light. 3) The reagents, consumables and containers used in the experiment are free of RNase contamination. 4) It is recommended to use linearized DNA templates for transcription. 5) When using modified nucleotides instead of wild-type nucleotides, the final concentration of the reaction remains unchanged. 6) If PCR products are used as transcription start templates, the amount of DNA template can be reduced by half. 7) Since the concentration of 10×Transcription Buffer is high, the high salt environment will cause the polymerase to be inactivated. At the same time, the buffer contains components that will form a precipitate with the template DNA. When preparing the reaction solution, it is necessary to adjust the order of adding components and calculate the system. First add water, then add buffer, NTP, and finally add template and enzyme to prevent the high concentration of 10× salt ions from affecting the enzyme. 8) In the purification of mRNA after transcription, wash the mRNA precipitate once with pre-cooled 75% ethanol. Repeated washing will affect the fluorescent group. Documents: Safety Data Sheet 10689_MSDS_HB250613.pdf Manuals 10689_Manual_Ver.EN20250613.pdf
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