Sometime my Sanger sequencing is longer than the amplicon seen on the gel. Have you meet the problem? What can cause this?
Could you post a copy of your chromatogram?
When I've seen this issue in the past, it's usually when the signal-to-noise ratio is low, and the base calling software erroneously makes base calls off of elevated background noise. Any chance you have a contaminant migrating with your sample?
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
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
BLAST primer, ipress, MFEprimer ? which one do you use?
31 December 2016 2,476 0 View
I have reverse sequences (AB1 format), can I base on reverse DNA sequences to perform nucleotide alignment, convert nucleotides to amino acids and deposit the sequence in GenBank database?
11 August 2024 5,138 1 View
I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...
10 August 2024 7,480 3 View
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 have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...
06 August 2024 3,130 2 View
I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...
05 August 2024 9,798 3 View
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
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
We are working on biopolymeric hydrogels. Our system is highly viscous and sticky, and the gel formed are high in strength. We are unable to use pH electrode and pH strip. Please suggest an easy...
30 July 2024 942 2 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