By Henrik Nielsen (auth.), Henrik Nielsen (eds.)
Recent perception into the transcripts generated from the mammalian genome (i.e. the transcriptome) has printed that transcription is a much more advanced phenomenon than formerly suggestion. In RNA: equipment and Protocols, professional researchers give you the approaches and techniques used to explain the constitution of messenger RNAs and non-coding RNAs which are transcribed by means of RNA polymerase II because the quick gene items in mammalian cells. keen on the constitution of the RNA items of “gene X” and the mapping of proteins linked to those RNAs, the quantity provides acceptable info for non-specialists in RNA biology. Written within the hugely winning tools in Molecular Biology™ sequence layout, many chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effectively reproducible laboratory protocols, and key pointers on troubleshooting and fending off recognized pitfalls. accomplished and sensible, RNA: tools and Protocols perspectives the transcriptional panorama with an appreciation for the function that proteins play within the processing and interpretation of genetic details in an try to extra our an important wisdom of the numerous items and complex regulatory networks that end result from it.
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Example text
PCR Templates for In Vitro Transcription 1. Design the oligos for PCR-amplification. 2. Make a standard PCR reaction. 3. Purify the PCR product using a commercial PCR clean-up kit (GenEluteTM PCR Clean-Up Kit Sigma) according to the manufacturer’s instructions. 2. 1. e. Unlabelled) Transcripts 1. 5 μL DEPC-treated dH2 O – 1 μL of template DNA (linearized plasmid or PCR-product) – 1 μL 10 U of the appropriate (in this case T7) RNA polymerase (see Note 9) – Incubate for 30–60 min at 37◦ C. 2. In Vitro Transcription of 32 P-Labelled Transcripts (see Note 5 for 32 P-Handling) 1.
2. Three different types of DNA templates for in vitro transcription. In the upper panel, a circular plasmid with the insert of interest cloned between a T7 promoter and a unique restriction enzyme site is linearized and transcribed from the promoter to yield multiple RNA transcripts terminated by “running-off” the template. In the middle panel, a DNA template (genomic DNA, cDNA, or a cloned fragment) acts as a template in PCR with a 5 -primer containing a T7 promoter (with no complementarity to the template) fused to a specific sequence complementary to the sequence of interest and a similarly specific 3 -primer.
Materials 1. 100 μM stock of biotinylated LNA oligo(T) capture probe (Exiqon) (see Note 1). 2. 5% (w/v) sodium N-lauroyl sarcosinate (see Note 2). 3. 1% (w/v) sodium N-lauroyl sarcosinate. 4. 1% (w/v) sodium N-lauroyl sarcosinate (see Note 3). 5. 5. 6. Quartz sand, baked at 220◦ C for 12 h. 7. Pestle. 8. g. Roche). 9. g. the PickPen system from BioNobile). 10. Yeast tRNA, diluted to 1 μg/μL in TE-buffer. 11. Thermomixer (Eppendorf). 12. g. from Ambion, ABI). 13. 5. 14. Glycogen carrier, 5 mg/mL (Ambion, ABI).