use a RNA fragment to construct a standard curve for a RT-PCR?

Can I use a synthesised RNA fragment containing the amplification target to construct a standard curve for a one-step RT-PCR? Can I also use the RNA fragment as the amplification control? What...

03 April 2019 3,858 5 View

Sanger sequencing is longer than amplicon?

Sometime my Sanger sequencing is longer than the amplicon seen on the gel. Have you meet the problem? What can cause this?

01 February 2019 781 2 View

Sequence LNA PCR product?

I am doing genotyping PCR. The probe has 3 LNA nt. Can I do regular Sanger sequencing to the PCR product to verify the genotype?

10 November 2018 9,433 0 View

A short amplicon got a long sequence by Sanger sequencing?

By PCR I got a ~500 bp amplicon from a microorganism. I did gel purification and sent for Sanger sequencing with the PCR primers from both ends. Now the sequencing result from the forward primer...

05 June 2018 3,014 3 View

Primer and probe TM of one-tube real-time nested PCR?

I am designing a one-tube nested real-time PCR. Only one probe will be used and the signal will be only collected from the second stage. Any suggestions for primer and probe TM design?

04 May 2018 7,274 5 View

One primer specific?

I am designing a Taqman PCR. However, I only can find a small continue specific fragment (9bp) in the target region. How should I design primers and probe? 1. place the 9 bp in either forward...

04 May 2018 8,728 6 View

QPCR amplicon did not have the band on the agrose gel

I did a QPCR, which gave me the CT around 20 and it had a sharp peak around 80 degree according to the melting curve. Then, I realized I need to extract DNA from it. So, I ran i5 ul of the 25 ul...

07 August 2017 8,496 3 View

Dose SYBR Green change amplicon running speed in an agrose gel ?

07 August 2017 4,839 1 View

Forward sequencing and reverse sequencing have different results

I have a ~700 PCR amplicon sequenced from both directions. However, forward sequencing and reverse sequencing have different results. The forward one has almost perfect matches with existing...

06 July 2017 6,790 4 View

How to find a genus or species specific gene for diagnosis?

For example, I know a bacterial genome. How should I search the genome with all bacterial genomes to find out a specific segment which can be used in PCR to diagnose the presence of the bacterium...

31 December 2016 3,912 4 View

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 4 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

How to calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 View

Anybody with an idea on how do design specific primers for detecting tomato resistance genes tm-1, TM-2 and Tm2^2?

I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There...

28 February 2021 606 3 View

What is the ideal efficiency for a qPCR?

hello everyone, I need to do standard curves for my qPCR, what is the ideal efficiency range? I tried a primer (Mglu2 receptor) that gave an efficiency of 90.2%. Is it accepted?

28 February 2021 1,254 3 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

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

Genescan gives 3 peaks instead of one?

Hi all, I am doing a genescan analysis for a deleted exon in the patient sample. The amplified region is 215 bp. In the patient sample because of homozygous deletion, no peak is observed while in...

23 February 2021 5,645 1 View

QPCR reaction efficiency with odd results what does this indicate?

I am working on a CFX 96 machine to determine reaction efficiency of my gene of interest. After performing a serial dilution of my cDNA template, I was able to find great reaction efficiencies for...

23 February 2021 809 3 View