I want to extract rotavirus RNA from feces of cattle
Hello Reham Karam,
Follow the steps below for extracting best quality RNA from stool.
1. Suspend 200 mg of stool in 600 ul of 0.9% NaCl containing 20 uMolar beta mercapto-ethanol. ( This is the maximum recommended
2. Mix it by pipetting gently, centrifuge at 10000 rpm for 5 minutes.
3. Collect the supernatant and discard the pellet.
4. Filter the solution in 0.22 um syringe filter.
Follow the QIAGEN protocol next https://www.qiagen.com/in/resources/resourcedetail?id=15aa2b1f-6047-4e9d-9d68-59ea58e248dd&lang=en
Hope it will help you
Biswaranjan
thank you for your valuable help
will i need any extra steps before cDNA synthesis
i read about the so called hydroxyapatite
will i help ??
If you are not going for sequencing (NGS) of the total RNA , hydroxyapatite method of rRNA degradation and cDNA normalization is not needed.
thanks alot
i aim for a sucessful PCR to head forward to seq so i should add hydroxy apatite
I have two bottles of cell culture media MEM Hanks and 199 Earls I dont need them now, but i will later. How can I preserve them for about 6 months , without loss in nutrients or contamination ?
04 May 2018 4,865 5 View
I am working on bovine rotavirus in Egypt, i searched and found little number of papers doing RT-PCR in Egypt What are the parameters that make me choose the correct one? the RNA nature of the...
05 June 2016 2,621 15 View
How much T.C media should I put in the 24 well plate or the 96 well plate or the roux bottle? Should I put the microlites of cells in each vessel? I am working on BHK -21 cells.
03 April 2014 6,564 1 View
Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...
11 August 2024 7,082 3 View
I have been doing the m6A dot blot for a while with no improvement, I am extracting the RNA, and I can see the dots although the three biological replicas give a different reading on the memberan...
10 August 2024 8,539 5 View
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...
08 August 2024 4,645 2 View
I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method...
08 August 2024 9,882 2 View
After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...
08 August 2024 1,668 3 View
I am currently working on LncRNA; to know the lncRNA-protein interactions I want to do RNA pull down assay, so I need to design primers with T7 promoter. I need assistance in this regard.
07 August 2024 6,622 1 View
I would like to understand potential safety concerns while handling SEB in the lab. Especially while working in animal house facility. Would like to know precautions for handling. Sigma MSDS...
07 August 2024 6,034 3 View
Recently, we observed that 99% of the sequences in our RNA-seq data corresponded to the E. coli genome. Despite multiple DNAse treatments after RNA extraction and ribosomal depletion, we were...
06 August 2024 807 3 View
I am planning to collect human fecal samples for metatranscriptomic analysis using MGI. These samples are from indigenous people living in a region with high temperatures. I will have access to a...
06 August 2024 1,367 3 View
i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...
05 August 2024 8,835 1 View