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Questions related from Moein Iranmanesh
Gene Knockout efficiency ? Hi everyone ! 'I've been working on a gene knockout in a Bacterial. The KO efficiency was explored for the first time after the limiting dilution and cell cloning by...
31 May 2025 6,155 2 View
Why is the concentration of plasmid DNA low after DNA? Plasmid purification is done by alkaline method. Can anyone suggest a better protocol?
31 May 2025 8,614 6 View
Hi everyone! After thinking about it on my own, I'm posting a question here. Has anyone ever tried creating qPCR standard curve samples (plasmid or DNA standard) by inserting multiple target...
31 May 2025 6,645 2 View
Problems with Mutation screening? We used pRE112 suicide plasmids to transfer the DNA fragment in the E.coli chromosome (as the recipient strain) to apply biparental mating through two screen...
31 May 2025 6,199 0 View
Does anybody can share an expression vector containing an endolysin for Gram-negative bacteria and a thermonuclease? We are looking for an expression vector co-expressing an endolysin against...
28 May 2025 6,011 0 View
What is best method for isolation bacteriophages, for Bacillus? Recently, The isolation of bacteriophages from sewage water is so difficult, How to obtain phages plaques when i make isolation...
28 May 2025 3,754 1 View
What is the protocol for the digestion restriction enzyme of phage? For example, what marker should be used, etc.?
28 May 2025 3,936 2 View
Can someone introduce a vector that simultaneously introduces two or more antibiotic resistances (markers) for better screening of resistant bacteria that lack the vector?
28 May 2025 3,532 4 View
Have you ever seen a small number of cells, 50-100 thousand, after centrifugation as a dry pellet in an Eppendorf tube without a microscope? Are there methods to potentially stain the cells in the...
22 May 2025 1,738 0 View
I'm doing a Western blot and haven't seen any protein for two months. After transferring, the marker is visible, but when I detect the protein once, wash it, and try to detect another protein the...
22 May 2025 589 3 View
I've pelleted down the overnight ampicillin supplemented cultures of DH5ALPHA for pET22b isolation 5 days ago and stored them on -20°C without adding any buffer or glycerol. Would the cells be...
09 November 2024 1,520 1 View
I have a plasmid. This plasmid has 2 promoters, the first one is T7P to express araC protein. And the second promoter is pBAD to express lacZ protein. Both promoters are on the same plasmid and in...
05 November 2024 7,408 2 View
I am getting errors. Either positive control cq values will vary or there will be no amolification. The knockdown genes are showing increased expression as per the amplification data. Can anyone...
05 November 2024 8,191 0 View
How to store SDS-PAGE for fluorescent imaging without fixatives? I am running protein samples on SDS-PAGE that are tagged with fluorophores. If I place the gel in a destain solution of...
30 June 2024 9,223 1 View
The source of classification bias in marker gene metagenome sequencing? a variability in the taxonomy classification of microbial communities when using different primer pairs (e.g. for 16S rDNA)...
30 June 2024 1,124 1 View
What is the reason or importance of diluting an enzyme before an experiment? I'm writing a report on enzyme activity and characterisation and i need help with understanding this: we were told to...
07 June 2024 8,806 2 View
Is a 5` phosphate needed for homologous recombination? I am a bit confused whether phosphorylated DNA is needed to achieve efficient homologous recombination in e coli. I believe there are methods...
07 June 2024 7,123 1 View
I am doing a CRISPR targeting to knock out one intron of my gene of interest in a cancer cell line and replace that intron with GFP. I am wondering if I should pick single cell clone with possible...
18 March 2024 9,783 1 View
I am amplifying target sequence 450 bp. I get single sharp band in the control and faint in sample with another sharp nonspecific product. why? I need to get one single band from my sample to...
18 March 2024 882 3 View
So, here is what I am dealing with: The Tm of the primers is 55 for the forward one and 56 for the reverse one. In -silico PCR produces 750 BP band, but in reality I get 3 bands. The third one...
18 March 2024 9,983 5 View
For example, If a PCR primer can detect ~100 fg of DNA, how we will calculate it to Bacterial copy number?
18 March 2024 1,886 2 View
I could not find a study that used CRISPR-mediated HDR for stable integration of large genes. I would be grateful if anyone could point me in the right direction.
06 August 2023 541 2 View
How can the design and synthesis of guide RNA molecules be optimized for improved CRISPR-Cas9 gene editing?
06 August 2023 1,442 2 View
What are the key challenges we face when developing a bacterial diagnostic kit based on CRISPR-Cas in a laboratory setting, and how can these challenges be addressed?
16 June 2023 7,450 1 View
I am thinking of inserting a small marker peptide into HDR fragments to detect and visualize CRISPR recombinant colonies. Is this possible? please guide me.
01 January 1970 9,596 2 View
I am trying to do a CRISPR/Cas9 knock-in and am wondering about the design of the donor vector (with the HDR template). I've been looking through many publications, but never actually find an...
01 January 1970 8,472 1 View
01 January 1970 2,583 1 View
I need step by step instructions to build 16SrRNA phylogenetic tree using software or website.
01 January 1970 4,843 3 View
I have insert having flanking regions with XbaI site and and a vector digested with XbaI. How can I prevent self ligation in insert as well as vector before ligation?
01 January 1970 9,459 2 View
Hello, Last week I asked my PCR results and I send Sanger seq. Well, there is one colony including mutation, I guess. How to cleaning my sample for Sanger?
01 January 1970 7,603 2 View
CRISPR-Cas13 can be used to detect and treat antibiotic resistance in bacteria. Is CRISPR-Cas13 technology more advanced than CRISPR-Cas9? Is CRISPR-Cas13 safer and more accurate than CRISPR-Cas9?
01 January 1970 1,433 0 View
I isolate RNA from Gram-positive bacteria using Trizol reagent (Invitrogen). I would like to know why the centrifugation speed should be reduced from 12000 g to 7500 g during the ethanol wash...
01 January 1970 7,478 1 View
What are the suitable software of designing primers for RCA method (rolling circle amplification)? Is the software and designing rules different from primer design for PCR?
01 January 1970 6,138 1 View
I've been trying to isolate a low copy plasmid from Enterobacteriaceae that has selective markers (Kanamycin). However, when I try to recover the plasmid, I can't get any positive bacterial clones...
01 January 1970 3,111 2 View
I'm facing challenges with the purity of plasmid DNA extracted from shigella for PCR applications. Despite using a commercial DNA extraction kit, I suspect the presence of contaminants or...
01 January 1970 5,554 2 View
My problem: sequencing result is low-quaility and dirty. Most sequence is matched with expected one, but there are some sites having two bases' signal, and the overall base signal is not strong,...
01 January 1970 1,325 1 View
I am trying to design sgRNAs for dCas9 fluorescent tagging, but all of the tools I find online are for standard CRISPR knocking in/out. Anyone have a recommended tool for dCas9?
01 January 1970 8,563 1 View
01 January 1970 5,260 0 View
I am to design sgRNA for Cas9 fluorescent tagging, but all of the tools I find online are for standard CRISPR knocking in/out. Anyone have a recommended tool for dCas9?
01 January 1970 8,443 0 View
I have a donor template with left and right homology arms (each 130bp long) designed for a specific sgRNA cutting site but now I want to use another sgRNA cutting 15 bp downstream of the original...
01 January 1970 4,960 1 View
Can anyone please let me know how to design Fluorophore-quencher Labeled Single-Strand DNA(FQ-ssDNA) probe for CRISPR Cas12a? Thank you.
01 January 1970 2,868 0 View
To design a specific primer for circular RNAs, are there any special conditions compared to the normal primer design? what is your suggestion?
01 January 1970 6,198 1 View
What concentration of antibiotic should I use to detect single CRISPR Knock out colonies?
01 January 1970 4,959 4 View
Is there any difference between the protein extraction protocol from frozen cells and cultured cells?
01 January 1970 4,168 3 View
1. My overexpression plasmid (empty vector) is 8200 bp. But it is showing 4000-5000 bp always in the agarose gel, which might be for the supercoiled conformation. But this large difference is...
01 January 1970 9,964 1 View
I would like a buffer (a standard, commercially available one would be best) for protein solubilization (non-denaturing) so protein IP is possible, which is also compatible with PCR, so preferably...
01 January 1970 1,376 0 View
In particular, can you explain why a gene is more highly expressed in PCR without reverse transcriptase than in RT-PCR?
01 January 1970 8,449 2 View
To produce a CRISPR Knock out in bacteria where my HDR (single-stranded and double-stranded) has the right homology arm of the left arm pair of 150, should HDR concentration be used for knocking?
01 January 1970 2,941 0 View
* In some of the papers of Pseudomonas isolates it is seen that bacteria that are susceptible to antibiotics are forming strong biofilms. What could be the reason? * Bacteria that are MDR show...
01 January 1970 6,147 0 View
The best online resource for conducting in silico PCR simulations of bacterial DNA.
01 January 1970 1,933 2 View
How to calculate the precision for a RT PCR assay?
01 January 1970 2,844 2 View
How is this done? "The reactivity of peptides was confirmed using western blotting and enzyme-linked immunosorbent assay (ELISA) assays"
01 January 1970 8,630 1 View
Hello! Can you give me some tips how to do CRISPR-Cas9 more effectively? Maybe you can give me some advice on this topic. This technology is new for me, I tried to conduct the experiments to...
01 January 1970 8,778 1 View
I want to find the UTR sequence of mRNA sequence of bacteria protein. Can anyone suggest a insilico process for that
01 January 1970 2,255 3 View
Can somebody share a standardised protocol for isolating phages against gram-positive bacteria
01 January 1970 9,472 1 View
Looking for a vector CRISPR-Cas9 containing sequence to delete a genes using gene specific gRNA.
01 January 1970 6,545 0 View
I plan to explore the integration of computational biology and artificial intelligence (AI) with laboratory and experimental work, including animal models, cell culture, clinical trials, and...
01 January 1970 5,701 4 View
I need your help with optimazing my PCR reaction. My PCR product should be 850bp. Starters Tm is 65, they do not form dimers or secondary structures and they’re specific (Blast doesn’t show any...
01 January 1970 1,317 3 View
Can I apply heat shock transformation method for Gram positive competent Bacteria? I need to transform a plasmid of 10 kbp in Gram positive competent Bacterial. Right now, I do not have...
01 January 1970 5,486 3 View
cDNA synthesis from RNA with reverse transcriptase reaction is a simple method and then gene expression will be checked with qPCR. After cDNA synthesis, we can store it at +4 degrees for a short...
01 January 1970 3,700 2 View
Why the 16s does not allow absolute quantification of microbial in microbiology? I have recently been given a microbial sequencing dataset based on the 16s method, but I have found that this...
01 January 1970 247 2 View
The last couple of months I've been trying to replicate a small part of the DNA by using PCR. While everything was working great at first the PCR just stopped working one day. I haven't changed...
01 January 1970 4,814 3 View
Hello experts in genetic modification, according to your experiences, do 2 mm and 4 mm electroporation cuvette have any differences in performance? or they just different in volume
01 January 1970 1,630 1 View
Why does BLAST results give 100% similarity to two bacterial species and only identified to the genus level? I did BLAST of the two cellulolytic species and one lignolytic species of bacteria...
01 January 1970 3,023 2 View
I run PCR to checked presence of specific gene in shigella but I received 2 bands, one band showed product size expected and another one showed higher product size. I would like to check the my...
01 January 1970 10,008 1 View
Do you know simle protocol of conversion ssDNA to dsDNA (applied to ancient DNA)? Without using commercial kits.
01 January 1970 6,908 1 View
What could be the reasons of protein not expressing in Bacillus Subtilis and Bt?? using vectors with autoinducibe promoters(Pylb and cyt1). I have performed SDS of 24, 48, 72, 96 and 120 hours. No...
01 January 1970 2,494 1 View
meone can help me with error prone PCR? How can you regulate the number of random inserted mutations in error prone PCR?
01 January 1970 1,592 2 View
I have a primer pair sequence for bacterial DNA. How to confirm the specificity of these primer pairs for their target genus? is there website to validate the specificity? I tried BLAST but...
01 January 1970 5,919 5 View
Is there a way to map gene expression data from real-time PCR on gene trees? I am trying to understand how the expression of paralogues changed over time. I have real-time PCR data and gene tree....
01 January 1970 8,304 1 View
I want to know the completeness and contamination level of bacterial genome sequence. I have the assembled FASTA file of the bacterial genome sequence. Are there any online tools where I can...
01 January 1970 8,466 1 View
Hello, I want to order Midiprep plasmid isolation kit, but I can't decide which brand to use. Can you recommend the brands you always use and think are highly effective?
01 January 1970 246 3 View
I noticed some white growth in the phosphate buffer that was prepared just a week ago. What could be the cause of this, and how can it be resolved? The buffer wasn't filtered.. Could this be the...
01 January 1970 259 1 View
I am currently doing In vitro transcription of two different RNAs, one is ~90bp size and another one ~200bp. I am using 2μg of DNA template, the reaction is kept for 3 hours at 37 degree. After...
01 January 1970 9,673 1 View
Has anyone ever tried creating qPCR standard curve samples (plasmid or DNA standard) by inserting multiple target sequences into a single template and then conducting experiments?
01 January 1970 7,133 1 View
I have synthesized a gene specific primer for amplification of my target gene. However, I am getting amplicon of only 100 bp rather than 210 bp (target amplicon size). So far I have tested so many...
01 January 1970 6,822 3 View
We are profiling RNA from soil samples and are wondering what others use for library preparation for metatranscriptomics? Any recommendations? What is your favorite kit and why?
01 January 1970 7,567 0 View
I need to precipitate extracellular protein from supernatant. A bit confused with ammonium sulfate calculation. Should I be adding: 1. 65g of ammonium sulfate in 100 ml of supernatant? OR 2....
01 January 1970 4,668 1 View
