What do I have to consider to design a long range multiplex PCR?

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Does anyone know a multiplex PCR with more than 5000 bp amplicon or a paper where primers for this are described?

I'd like to describe human genomic DNA quality from different starting biological material.

06 July 2012 6,584 1 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Can thermal cycler machines be problematic during PCR?

Hi, my question is about the heating of thermal cycler machines and I hope some of you had experienced a similar thing previously. There are two thermal cycler machines in the lab(BioRad) and for...

26 February 2021 4,777 4 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 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

Yousry Hawash

First, you have to define your target gene(s), primers, They have to be highly specific for the organism, primers have to be work in similar thermal conditions to be easily multiplexed.

Navonil De Sarkar

Try to keep more or less similar amplcon (and again not exactly the same). Then you can manege the extension time. Use Long PCR high fidelity taq, and obviously the comments Yousry has made follow those...

Bellmunt Elena

Thank you very much for your comments!