Opinions Requested Please

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Opinions Requested Please

Postby mbgeek16 » Nov 29 2016 6:52 am

I have been given a 12 week time frame to complete two kinds of project (below). Are 12 weeks a reasonable amount of time to complete the projects if following this protocol? Any comments on the time frame and the appropriateness of the protocol for the tasks would be greatly appreciated.

Project descriptions:

(1) Purify two dsRNAs of expected sizes from Rhizoctonia solani, cDNA clone and sequence analyze.

(2) Assemble, annotate and determine markers and phylogenetic relationships of two R. solani mitochondrial genomes with related fungi and reported R. solani mitochondrial genomes.

Project 1 summary:

A. Make full-length clones of the two large (~25kb and ~23kb, exact size unknown) dsRNAs (L1 and L2), the 2.4 Kb (S1) and 1.2 kb (S2) dsRNAs from 1A1 Rhizoctonia solani strain following the protocol below. No sequence information is available for any of the dsRNAs.
B. Assemble full-length sequence of the above four dsRNAs
C. Analyze all sequences bioinformatically including gene finding and annotations.
D. Present your results in Figures and Tables.

Protocol provided:

1.Extract and gel purify dsRNA (separate protocol, not attached) band of the expected size(s) using both 1kb DNA ladder and a high MW DNA marker.
2. Poly A tail with E. coli poly(A) polymerase
3. Make cDNA with oligodt + random hexamer and Superscript II or III Reverse Transcriptase (Life Technologies).
4. Treat with RNaseH to remove RNA.
5. Anneal the cDNA strands to make partially double-stranded DNA molecule:
(10 min at 80degreeC, for 16h at 65degreeC and for 3 H at 30degreeC.
6. Fill in the resulting hybrid with Klenow Fragment.
7. Ligate with given Not1 linker.
8. Blunt end ligate in a plasmid vector in Sma1 site (pBR322 or Lambda dash vector for large dsRNA, pGem3Z for small dsRNA)
9. Clone and screen by miniprep, by Not-1 restriction digestion and gel electrophoresis to isolate the cDNA insert.
10. Estimate the size of the insert from the gel
(Restriction map and sub-clone if necessary with EcoR1 and Xba I)
11. Sequence and align.
12. Make complete contig of dsRNA, analyze for ORFs, show homology, if any to NCBI accessions.
13. Make a final report.

Project 2 summary: NGS analysis

A. Prepare a Circular map of two Rhizoctonia solani mitochondrial (mt) genomes (from paired reads of ~26,000,000KB in total size) showing the genes, their position, and on which strand they belong.
B. Find genes with gene names, Open Reading Frames (amino acids sequences),transposons, and introns, etc.
C. Present your results in Figures and Tables.
D. Compare two given mt genomes at the nucleotide level and find selected AG-specific SNPs or INDELs that can be used for rapid diagnostics.
E. Determine the phylogenetic relationships of two mitochondrial genomes with related fungi and reported R. solani mitochondrial genomes.

Please let me know if you need any additional information.
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