By Andreas Werner
Animal Endo-SiRNAs: tools and Protocols offers various ways to enquire endo-siRNAs. those comprise protocols appropriate to review brief RNAs expressed at a low point and version structures which are rather appropriate to enquire particular points of endo-siRNAs, their synthesis, their genomics or regulatory position. Written within the hugely winning Methods in Molecular Biology series layout, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols and pointers on troubleshooting and averting recognized pitfalls.
Authoritative and functional, Animal Endo-SiRNAs: tools and Protocols includes functional information which are absent in general lab manuals.
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Additional resources for Animal Endo-SiRNAs: Methods and Protocols
The specific brand of Taq polymerase is not important. However, we recommend using a hot-start enzyme with high fidelity since nonspecific amplification products may adversely affect sequencing and polymerase mistakes can interfere with adapter trimming and read mapping during data analysis. 7. You can clone the PCR products with the help of a standard blunt-end cloning kit (for example the pJet system from Fermentas) and sequence the inserts from 5 to 10 colonies by standard Sanger sequencing for quality control purposes.
It is important to consider how many samples are going to be pooled and choose the indexes accordingly. Since the barcode sequences will be of low complexity, a barcode combination should be chosen that will have at least one A or C and one G or T at each position (see legend to Fig. 2). Especially if only two libraries are combined (for example a small-scale experiment run on a MiSeq system) de-multiplexing may prove challenging if a unsuitable index combination was used. We indicate suitable combinations of the barcodes described in this protocol in Fig.
2 Oxidation of Vicinal HydroxylGroups 1. 75 μL of NaIO4 solution (200 mM) (see Note 1) Incubate the mixture for 30 min at room temperature. Then add 3 μL of 100 % glycerol to quench the remaining NaIO4 and incubate for 10 min at room temperature. 2. 5 μL of 2 M NaOH solution to the reaction mixture and incubate for 90 min at 45 °C (see Note 2). 3. Purification of modified RNA: Purification of the modified RNA is necessary at this point (with- or without β-elimination) because the SDS in the sample leads to an insoluble precipitate 36 Katharina Elmer et al.