Question

Asked 5th Aug, 2017

CJ CheilJedang

CDNA synthesis of norovirus

Hello

I tried to synthesize cDNA with norovirus.

After RNA extractiom, first of all, real time rt pcr was performed to calculate viral copy number. The high copy samples were processed with cDNA synthesis, and then the products were conducted conventional rt pcr to confirm cDNA.

The targeted band was confimed in PCR reaction with one step RP PCR kit (thermo)...but no band in respective reaction, superscipt reverse transcriptasd and taq polymerase (recently purchase)

Does anyone help me?

Royal Veterinary College

Paul Rutland

University College London

Katie A S Burnette

University of California, Riverside

Jack M Gallup

Vaxxinova/Newport Laboratories, Worthington, MN

Frederic Lepretre

Université de Lille

Sharmin Hasan

Sam Houston State University

Hi, I think your cDNA is fine but what primers are you using for PCR? Annealing temperature of primer is important for PCR success. I would suggest, check your primers annealing temperature and re-do your PCR with a different primer set. Best wishes!

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1 Recommendation

B. P. Ray

Bangladesh Institute of Nuclear Agriculture

Helo, i think you can try through gradient PCR by Q5 high ferdility Taq polymerage.

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Mohammed Abdulabbas Mlaghee

University of Al-Qadisiyah

Article Norovirus RNA Synthesis is Modulated by an Interaction Betwe...

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Any tips for RNA extraction from stool matrix using RNeasy Mini Kit?

Question

3 answers

Asked 8th May, 2023

Megan Arias

Product of interest: ZeptoMetrix Norovirus GI and GII External Run Controls. https://www.fishersci.com/shop/products/nattrol-gp-external-run-controls/21100555

I am trying to run the RNA extraction with the RNeasy Mini kit I currently have available to me. My problem is that I have never worked with a stool matrix and I am green enough to not fully understand the necessary adaptations to make this work. After looking around at other protocols for stool extractions, I decided to start with pro K digestion + Buffer RLT then following the kit the rest of the way. This failed. The nanodrop was nearly a straight line. I am working with a limited amount (there's only .5ml in a tube) so I'd love to hear other ideas and tips for RNA extractions from stool matrix using RNeasy Mini kit.

Side note: I'm using this kit successfully to extract RNA from oyster matrix post-pro K and skimmed milk flocculation.

Can someone explain to me why most surveillance studies studies such as norovirus epidemiologic stusies use large samples sizes?

Question

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Asked 9th Jul, 2020

Akongnwi Emmanuel Mugyia

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013–2015" in which you use a sample size of 484 patients.

Molecular epidemiology of norovirus associated with acute gastroenteritis in Taizhou, China: A retrospective study"

in which you use a sample size of 1464 patients.

How is it possible to detect Adenovirus when using an RNA extraction kit even though Adenovirus is a DNA virus?

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Erick Kipkirui

I have been doing some multiplex work with rotavirus, adenovirus, astrovirus norovirus GI/II.

Trouble with gene amplification using cDNA as a template :(

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Asked 5th Jun, 2017

Parul Singh

Hi everyone,

I am trying to clone a gene from induced fungus and after doing RT and using cDNA as a template for gene specific PCR, I noticed the expected band size but before going ahead when I ran a confirmatory PCR, the band disappeared completely and now after repeating several times, I just can't find it, not sure what went wrong? does it happen often or something sounds strange? I doubt RT gone bad? any help and suggestion is much appreciated!

Minor but critical steps when handling mixtures?

Question

4 answers

Asked 19th Apr, 2016

Chinkyu Lee

Hello,

I asked similar question previously.

And I appreciate all of those who answered my question about handling of cDNA.

I want to ask 2 questions.

1. Suppose components in mixture are evenly distributed well by voltexing and then spindown. If I conduct voltexing and spindown more, the components will still be distributed evenly without any difference compared to the state before additional voltexing and spindown?

No difference between voltexing and spindown just one time

and multiple times of voltexing and spindown when handling specific mixture?

2. The aim of spindown is to prevent loss of mixture due to partial mixture separated due to voltexing. This is what I know so far about spindown.

so If no such separated mixture is detected,

only voltexing is ok for mixing components evenly?

Thank you!

(PS : I heard that voltexing may be not good option for mixing because of

shearing problems but I have never experienced troubles so far. But I

just want to get confirmed about my two questions above.)

CDNA conversion kit (QuantiTect Reverse Transcription Kit-Qiagen) can be used for cloning purpose of Human gene?

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1 answer

Asked 24th Feb, 2017

Murugan Nandagopal

My purpose is to clone a human target gene (for eg:insulin gene) into a cell line and express the same in cell line for research purpose.

My plan is to take RNA from human tissue and convert it to cDNA,

and cloning the cDNA into a suitable vector and transfect the vector into the Cell line.

is it right way to do, if so, qiagen kit will transfer the RNA into the full length of cDNA which codes for that particular protein ?

CDNA dilution storage dilution step

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Asked 14th Mar, 2017

Marc Paul O'Sullivan

With some kits, you make up 20ul of cDNA, and you can store this cDNA at -20oC. Then in the PCR step, you are asked to dilute 5ul of this cDNA by 80X and are recommended not to store it at this diluted amount. Does anyone know why?

What should the concentration of cDNA of bacteria be to do qPCR?

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Asked 12th Mar, 2014

Surendra Osti

Qpcr

I am having difficulty in amplifying the 5' end of the cDNA. Is there any efficient way to do so?

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3 answers

Asked 22nd Dec, 2014

Abiskar Gyawali

This segment of the 5' end to be amplified is around 450bp. I have already used two sets of primer but till now what I have visualized is the primer dimers at the end for both primers. I had also done the PCR at different temperature gradients for annealing, still no good signals.

Vector-Capping: A Simple Method for Preparing a High-Quality Full-Length cDNA Library

Article

Jan 2005

Seishi Kato

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains...

First strand cDNA synthesis v1

Preprint

Full-text available