Why do the nonspecific bands present when using high template concentration in nested rt-pcr?

10 October 2018 0 6K Report

Dear kindness,

I had a problem that I found nonspecific bands in the second round of nested rt-pcr and the specific band was not sharp when using 2ul of the first round template. Then, I reduced the template concentration to 1ul, 0.5ul and 0.25ul the specific band was increased and sharper than using 2ul. How can explain this condition?

Thank you.

Badges
Science method

More Wiyada Dankai's questions See All

Can detect the gene methylation in normal cytology of cervical scarping?

Dear all, I try to detect the gene methylation in cervical cancer patients and abnormal cytology comparing with normal cytology of cervical scarping by Methylation specific PCR method. In...

02 March 2017 3,571 4 View

What is the best formula between 2^dCt and 2^ddCt for analyzing the data from stress and non-stress condition?

Dear, researchersI try to study the gene expression between stress and non-stress condition, for example, I treat the sample with H2O2 at 37C for 1 h (stress condition) while the normal condition...

09 October 2015 3,590 37 View

Can I use sodium nitrite (NaNO2) to be the nitrosative stress?

Dear all, I want to test the nitrosative stress response with the fungi. Can I use sodium nitrite (NaNO2) to be the nitrosative stress? Thank you all.

06 July 2015 8,499 4 View

What is the difference between BestKeeper and Genorm program?

I want to validate the reference gene in real-time PCR, and I want to analyse the cq data. I want to know how does the difference between BestKeeper and Genorm program, which one is the...

08 September 2014 9,324 0 View

Where does the 1 kb DNA Ladder come from?

I wan't to know about the original of 1 kb DNA Ladder. Where does the 1 kb DNA Ladder come from? Lambda DNA right or not? Thank you.

07 August 2014 4,676 3 View

Do you have suggestions of protein lysis methods for western blot?

Hello everyone, I lyse the cell culture (THP-1) with RIPA buffer but it interfere both the nano drop and bradford assay. Can I only use the water to lyse the cell?Thank you.

07 August 2014 9,595 18 View

What is the criterion to chose gene control in real-time PCR?

I want to know about the criterion to chose gene control between actin and beta-tubulin. Please suggest any criterion.

04 May 2014 1,563 7 View

How do you prepare the N9 agar for bacteria stress?

I want to prepare the N9 medium but I found only the N-9 broth formula.

04 May 2014 1,271 2 View

What's Homologous recombination in filamentous fungi?

I want this definition. What's the mechanism of this?

02 March 2014 4,896 0 View

What's the caution in real-time PCR?

Real-time PCR

02 March 2014 3,022 12 View

How to confirm the site-directed mutagenesis result without performing NGS?

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 can't see the ssDNA band after performing asymmetric PCR. Is there any way to do this?

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

How much total RNA concentration to be extracted from sorted plasma cells from bone marrow of C57BL/6 mice for RT-PCR ?

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

Does anyone have issues using Prepman Ultra reagent for MicroSeq ID bacterial, fungal and yeast sample preparation?

I have been attempting to extract DNA from Bacterial, Fungal and Yeast banked samples (>1e7 cells) using Prepman Ultra reagent and I seem to be struggling to obtain a sequence. Although the...

01 August 2024 2,079 0 View

PCR showing no bands - Master mix and primers didn't mix?

Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...

31 July 2024 2,406 6 View

Dimensions of an MJ Research 96-well alpha module?

Can anyone with an MJ research / BioRad PCR machine from ~2010 or earlier tell me the external measurements (LxWxH) of the removable standard PCR alpha modules that can be removed from a PTC200 or...

30 July 2024 2,867 0 View

How to retain amplicon yield after Ampure bead cleanup, following a 2-stage PCR protocol?

Hello all, I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) -...

30 July 2024 841 2 View

Someone have the key or the installer of the program Mx pro 3000 (PCR real time)?

I need to install this program

25 July 2024 4,756 0 View

Illustra™ MicroSpin™ G-25 columns what it is used for?

Hello, We found three packages of Illustra™ MicroSpin™ G-25 columns in the cabinet of an unused lab. They are very old but have never been opened. I have never used this kit before, and I couldn't...

25 July 2024 4,927 3 View

How do I run a dead well test for a QuantStudio 5 qPCR machine?

I am preparing to run a lot of samples on a 384 well plate and I wanted to test if any well in my PCR machine is dead so as not to lose any data. I would really appreciate help with how I can do...

24 July 2024 8,172 1 View