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?
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
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'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
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 have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...
05 August 2024 8,835 1 View
A Markov-like Model for Patient Progression" Markov Chain Monte Carlo (MCMC) Markov Chain Monte Carlo (MCMC) is a powerful computational technique used to draw samples from a probability...
05 August 2024 10,079 0 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
I am performing ligation of the plasmid and a target gene. The steps I have taken are: 1. Double digestion of the plasmid and target gene 2. Ligation of the plasmid with the target gene 3....
05 August 2024 2,570 3 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
Read the journal article by Douglas M. Lambert, “The Eight Essential Supply Chain Management Processes,” Supply Chain Management Review, Vol. 8, No. 6 (2004), pp. 18-26
04 August 2024 9,919 4 View
How does the atmosphere affect the flow of matter and energy on Earth and flow of energy in the biosphere related to the flow of food through a food chain?
02 August 2024 9,644 0 View