09 November 2024 1 2K Report

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 viable for mannual plasmid isolation?

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Marker gene metagenome sequencing?

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)...

29 June 2024 1,048 1 View

fluorescent imaging without fixatives?

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...

29 June 2024 8,720 1 View

Is a 5` phosphate needed for homologous recombination?

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...

06 June 2024 7,065 1 View

Diluting an enzyme before an experiment?

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...

06 June 2024 8,714 2 View

How do you get Rid of Nonspecific Bands in PCR?

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...

17 March 2024 9,914 5 View

PCR nonspecific product?

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...

17 March 2024 804 3 View

CRISPR targeting in cell line, flow or single clone?

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...

17 March 2024 9,713 1 View

How convert Bacterial DNA concentration to copy numbers?

For example, If a PCR primer can detect ~100 fg of DNA, how we will calculate it to Bacterial copy number?

17 March 2024 1,816 2 View

Guide RNA for improved CRISPR-Cas9 gene editing ?

How can the design and synthesis of guide RNA molecules be optimized for improved CRISPR-Cas9 gene editing?

05 August 2023 1,375 2 View

Can it be used for large gene production (4 kbp) via CRISPR-mediated HDR?

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.

05 August 2023 486 2 View

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Is there a problem with my RNA pellet?

Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...

11 August 2024 7,082 3 View

How to confirm the site-directed mutagenesis result without performing NGS?

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

How does grain and grain boundary affect the ceramic when studying its dielectric properties?

I am not able to get good literature and the physics behind how first these grains and grain boundaries arises out of no where when we make a pellet to study its dielectric properties and then how...

07 August 2024 5,177 3 View

AMAXA Lonza nucleofection lower volume of buffer?

Does anyone tried to do nucleofection with AMAXA by Lonza with lower than recommended amount of buffer in the cuvettes (100 ul)?

07 August 2024 4,616 0 View

Why I can't see any band in SDS-PAGE?

Currently, when I run SDS-PAGE, I don't see any bands at all, even though I used the same material just a day ago and it worked fine.... In our lab, we dilute the 10X running buffer to 1X and...

06 August 2024 5,373 2 View

Where should the ARS sequence be introduced on a plasmid?

Hi all, I need to introduce an ARS (autonomously replicating sequence) in my plasmid but I'm not sure which position would be the best. Does anyone have any suggestion? A picture of the plasmid...

05 August 2024 1,573 4 View

Why after performing site directed mutagenesis ,I don't see any colony after transformation?

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

Why might the impedance values for DI water and 0.1X PBS buffer solution exhibit a decreasing and increasing trend, respectively over time (HP 4194A)?

Hello everyone, I'm encountering an issue with my electrochemical impedance spectroscopy (EIS) measurements and would appreciate some insights. Experimental Setup: Electrodes: Gold interdigitated...

05 August 2024 3,783 2 View

Why my colony PCR results of my recombinant bacterial not showing any results?

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

What is the best blank for nanodrop if I want to read a recombinant protein concentration?

Is it the "elution buffer" or the "dialysis buffer"? Note: I'll be using NanoDrop OneC

01 August 2024 967 3 View

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