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

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

How do you search primer specificity?

31 December 2016 2,432 0 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

How can I represent SDS-PAGE results for machine learning analysis?

I am trying to classify and analyze the results of an SDS-PAGE based array for bacterial detection using machine learning, but I have trouble finding the best way to represent the results with...

27 February 2021 9,176 3 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

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 my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 3 View

EMSA troubleshooting. Protein-DNA complex not migrating, cold probe lane with multiple bands. Can anyone help?

I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA...

24 February 2021 8,325 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

Errors in DNA sequences?

I am trying to better understand the scope of DNA replication and sequencing errors, e.g. 1. I have seen similar error rates of 10e4 to 10e5 for cell & instrumental DNA replication,...

24 February 2021 4,397 3 View