---+ in vitro RNA synthesis(T7 RNA Polymerase ) T7 RNA Polymerase is used for in vitro mRNA synthesis and is highly specific for the T7 phage promoter. T7 in vitro transcription kit is intended for the synthesis of large amounts of unlabeled or low specific activity RNA for a variety of uses, including in vitro translation, antisense/microinjection studies, and isolation of RNA binding proteins. ---+++ DNA Template Preparation Linearized plasmid DNA, PCR products or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with the T7 RNA Polymerase provided that they contain a double-stranded T7 promoter region upstream of the sequence to be transcribed. 1. Linearization of 3 ug of plasmid (obtained by Miniprep) with designed restriction enzyme. Incubation at 37ºC (Optimal temperature) for 3h. 1. Check linearization of plasmid on gel. Make ture got the linear plasmid in the expected size and no smear (perfect clean bands). 1. Extract DNA with an equal volume of 1:1 phenol/chloroform mixture, repeat if necessary or use zymogen DNA Clean & Concentrator kit . ---+++ in vitro transcription 1. Thaw the frozen reagents(Invitrogen™mMESSAGE mMACHINE™ T7 Transcription Kit or New England Biolabs™ HiScribe™ T7 Quick High Yield RNA Synthesis Kit). * Place the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not be frozen at –20°C. Vortex the 10X Reaction Buffer and the 2X NTP/CAP until they are completely in solution. Once thawed, store the ribonucleotides (2X NTP/CAP) on ice, but keep the 10X Reaction Buffer at room temperature while assembling the reaction. Thaw out all of your reagents on ice, and keep them on ice at all times. Do not speed up their heating. Do not take out the T7 RNA Polymerase enzyme until you need to add it to the reaction. * All reagents should be microfuged briefly before opening to prevent loss and/or contamination of material that may be present around the rim of the tube. 1. Assemble transcription reaction at room temp. Use filter tips for all pipetting. * The spermidine in the 10X Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice. * Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. The following amounts are for a single 20 µL reaction. Reactions may be scaled up or down if desired. * Component Amount * Nuclease-free or DEPC-Treated Water to 20 µL * 2X NTP/CAP 10 µL * 10X Reaction Buffer 2 µL * Linear template DNA 1 µg * 2X NTP/CAP 10 µL * Enzyme Mix 2 µL 1. Mix thoroughly. * Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube. 1. Incubate at 37°C for an amount of time that varies by your intended product's length: * More than 300 bp: 2 hours * Less than 300 bp: 4 - 16 hours 1. Check RNA quality on denaturing formaldehyde gels. Transfer into -70 freezer ℃. ---+++ Troubleshooting 1. Failed Transcription * DNA template not pure * Complete failure of the in vitro transcription reaction might be the result of using a poor-quality DNA template. DNA prepared with standard miniprep procedures should be of a sufficient quality for in vitro transcription. However, contaminants such as ethanol carried over from the DNA purification process can inhibit the RNA polymerases. Typically, precipitating the DNA template with ethanol and resuspending it will resolve the contamination issues. Contaminants like salts can inhibit the activity of the RNA polymerase. Desalt your template DNA by using a Clean-up kit. * RNase contamination * RNase contamination might be the culprit for the failed transcription. RNase can be carried over from the plasmid purification process or inadvertently introduced by inappropriate handling. RNase will degrade and RNA it comes in contact with. To prevent RNase from destroying your transcripts, use an RNase inhibitor in your transcription reactions. * Use an RNase inhibitor * Transcription templates that have been linearized incorrectly can result in transcription failure. If you are using a linearized template for transcription, verify that the sequence and restriction map is correct. 1.Incomplete Transcription * Incorrect linearized template * If you are using a linearized template, confirm the sequence and restriction sites. Unexpected cut sites can mean that the transcription template is shorter than expected. Try linearizing your template plasmid with a different enzyme. Confirm the sequence and restriction sites and check an aliquot of purified DNA on an agarose gel. *Degradation of RNA sample buffer * Multiple freeze-thaw cycles as well as the use of old buffers can affect the reaction. The RNA will not run on its true size. * The presence of cryptic phage RNA polymerase termination sites could be causing the transcription reaction to stop before the end of the template sequence. To resolve this, subclone your template into a different plasmid with a different RNA polymerase promoter. * Adjusting the transcription reaction conditions can resolve the problem of incomplete transcription. If the nucleotide concentration is too low, it might be limiting the reaction. Always use a nucleotide concentration of at least 12µM, and if you think this is the problem, you can increase the concentration to 20–50µM (Note: If you are synthesizing labeled transcripts, increasing the nucleotide concentration may require adding cold NTP, and this will decrease the specific activity of the final product). * If the template sequence is G/C rich, the transcription reaction might be terminating prematurely. For these templates, the amount of full-length transcript can be increased by decreasing the temperature of the transcription reaction *Reference* 1 https://www.thermofisher.com/order/catalog/product/AM1340#/AM1340 1 https://www.promegaconnections.com/in-vitro-transcription-common-causes-of-reaction-failure/ 1 https://zageno.com/l/ts-in-vitro-transcription 1 https://www.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050 -- Main.PengGeng - 09 Apr 2020
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Topic revision: r4 - 2020-12-03 - ElizabethRobinson