BLAST primer, ipress, MFEprimer ? which one do you use?
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 3,907 5 View
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 828 2 View
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 9,469 0 View
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 3,067 3 View
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 7,312 5 View
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 8,765 6 View
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 8,542 3 View
As mentioned in the title, has someone compared the running speeds between SYBR Green-DNA polymerase - amplicon and conventional polymerase -amplicon in an agrose gel?
07 August 2017 4,883 1 View
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 6,835 4 View
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 3,945 4 View
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
I am staining some brain sections stored in cryoprotectant that express a Histone H2B- GFP fusion protein that were generated ~10 years ago. I know I need to enhance signal with an anti-GFP...
07 August 2024 5,338 2 View
I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...
05 August 2024 9,059 3 View
How to design VN primer to attach with universal reverse primer
05 August 2024 2,116 3 View
It's an end-point PCR protocol. I'm using 1.5% agarose gel with SyBR Safe dye and TBE as a running buffer, visualization on BioRad XR+ system. I was primarily thinking of primer efficiency,...
01 August 2024 4,673 4 View
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
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
I want to introduce 2 mutations using Agilent's multi-site mutagenesis kit, I designed the primers using their online tool and it gave me 2 sets for primers (1 set for each mutation) so I was...
25 July 2024 6,517 3 View
Hi everyone, I have extracted DNA from a biogas bioreactor using Qiagen kit and prep cDNA library then used this library as template to optimize primers for qPCR (taken from papers). Some of the...
23 July 2024 1,329 5 View
Hi Everyone, I'm using an siRNA kit to knock down a target gene. The kit guarantees that the negative control doesn't target any sequence in mouse genome, and when I use BLAST I don't find any...
23 July 2024 2,673 6 View
