Can anyone suggest a solution to this problem with cDNA synthesis from an RNA virus (Agarose gel) using the Tetro cDNA synthesis kit?

Jahwarhar Izuan Abdul Rashid @Jahwarhar_Abdul_Rashid

02 February 2014 0 2K Report

Does anyone use the Tetro cDNA kit for cDNA synthesis? Recently, I have synthesized cDNA from an RNA virus using this commercial kit. I have followed their recommendation methods: Mix all of component kit gently by pipetting. Incubate samples at 45°C for 30 min. Terminate reaction by incubating at 85°C for 5 min, chill on ice.

Below is the picture of cDNA agarose gel. There are no signs that cDNA is generated based on band location at the high of kb size (1000 kb above) But there was a few single bands appearing at the lane 1,2,5,6 which is approximately below 50 kb in size (using a specific gene primer; 10 uM; 25 bp) meanwhile 3,4 -no band appeared using oligo Dt primer. Is it possible that my RNA is not fully transcribed to cDNA? Can anyone suggest the solution for my cDNA synthesis problem?

Badges
Science topic

More Jahwarhar Izuan Abdul Rashid's questions See All

It is possible that crude DNA/RNA extract (without PCR amplification) can be detected using a DNA sensor?

Is it any previous work claimed that their DNA biosensor capable detect the crude DNA/RNA extract without doing PCR amplification? Besides low DNA concentration problem, How they overcome the...

31 December 2014 6,708 0 View

How can the conductivity of an electrode enhance the sensitivity of target detection? Or are they not related?

Is it possible that when we have improved the conductivity of the electrode the sensitivity performance does not give significant changes from before optimizing the electrode?

04 May 2014 7,587 3 View

How might one verify the presence of amplicons ssDNA using Asymmetric PCR technique ?

When I visualised the band on the gel electrophoresis, there were not many different changes when compared to CDNA product using normal PCR technique in terms of the size of the band. But I...

01 February 2014 7,422 1 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

Ester cleavage conditions?

Hi all, I need to cleave the ester in Ethyl 2-amino-1H-imidazole-5-carboxylate to make the carboxylic acid and retrieve the product from solution to use in downstream applications of linking to...

01 March 2021 8,766 3 View

How vaccines of covid 19 work?

Millions ask how vaccines of covid 19 be successful and safe, its here to explain generally how vaccine of pfizer work ,mRNA vaccines teach our cells how to make a protein that will trigger an...

28 February 2021 2,723 4 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

24 February 2021 8,618 3 View

Recommendations on homogenizers for RNA isolation?

I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the...

24 February 2021 3,623 3 View