Questions tagged [lab-techniques]
Questions relating to protocols, procedures, and good practice when using laboratory equipment.
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Storage conditions for Propidum Monoazide
I'm a junoir research assistant working on a mini project under my supervisor right now.
I was just wondering if anyone else has worked with Propidium monoazide (PMA) in water for PCR. Currently our ...
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Neutralization Assay: Comparision of IC50 values between different viral loads
I've been performing Neutralization assays for HIV pseudovirus against a dilution series of different antibodies, and was wondering whether the viral load influences the measured IC50.
My specific ...
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How can I non-destructively blind/cover one drosophila eye for behavioural assays?
I am looking for a protocol for non-destructively covering one eye of Drosophila for a behavioural and subsequent imaging experiment I am planning. I am aware of using wax to paint over the entire eye ...
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What are ways to measure carbon fixation of microalgae?
So I am conducting a very simple experiment about microalgaes, particularly their biofixation of carbon dioxide. In a small compartment, where microalgaes are suspended in water with an air pump that ...
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Extremophile hunting and identifying unknown microbes
I'm going extremophile hunting! We're doing a trek by boat between some islands with semi-active volcanic terrain to try to document some interesting species, and running into a problem I've never ...
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How come SSBPs in RPA don't bind primers?
I've started reading about the recombinase polymerase amplification (RPA). I'm learning that in RPA, recombinase enzyme binds to primers, then makes them anneal to the complementary target DNA strand, ...
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Can single-Use Gloves carry bacteria on their outer surface?
In bacteriology, can I touch items (e.g. oxidase discs) with my gloves not fearing to contaminate these items?
Are these gloves (e.g. Disposable nitrile gloves) made so that bacteria can't adhere to ...
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Where can I find the standard curves for Bovine Serum Albumin (BSA) and ovalbumin as determined through the Bradford Assay?
I was wondering if anyone would please be able to point me in the direction of a database or something similar which contains standard curves/absorbance levels for BSA and ovalbumin, as measured using ...
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Immunoaffinity chromatography: avoiding damage to the antibodies from proteases
What are the possible methods to prevent the digestion of antibodies (mainly Polyclonal) by proteases during affinity chromatography?
I read some papers about doing modifications to the anitbodies:
...
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Methods of denaturing BL21 E.coli other than using lysis buffer and sonication
The goal is to try different denaturing methods for BL21 E.coli. However, finding detailed paper on this is challenging as most articles do not seem to include the details of their denaturing ...
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chip sequencing
I understand the concepts of steps in Chip-seq up to DNA purification, but I don't get how one can then amplify the purified DNA samples that are once bound to the proteins.... Since sequence-specific ...
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What are some experimental techniques to identify binding partners?
What experimental techniques can be used to identify binding partners for a given protein? I know that if you have some candidates that may bind to a given protein, then you can use techniques such as ...
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What nutrients are necessary for Bacillus subtilis germination?
What are the necessary conditions to germinate Bacillus subtilis spores? Under laboratory conditions, spores of B. subtilis are often heat-activated for 30 min at 70°C to increase their germination ...
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Primer design for site-directed mutagenesis
In our practical course about modern cloning methods, we performed point mutations on a promotor via site-directed mutagenesis.
As far as I understand that method you need forward and reverse primers ...
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Why is my tissue sample coagulating during centrifugation? How can I prevent this?
I am working on extracting water-soluble compounds from a biological tissue sample. A problem I am encountering is that the sample is too cloudy with debris/large tissue pieces to filter into HPLC ...
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How to prepare ethanol-preserved insects for imaging using SEM
I have aquatic insect larvae (soft-bodied) currently preserved in 70% ethanol. I would like to image these using a scanning electron microscope (SEM). I am wondering whether I need to/can start at ...
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What colors I am looking for in the TSA stain in this paper?
I'm just having a little bit of trouble and want to make sure that I am seeing the same things that the original researchers were seeing. This is the name of the paper Hair Cycle Control by Estrogens: ...
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Why is cDNA usually lacking in terminal sequences of the template mRNA?
It appears from presentations I have attended that cDNA often lacks terminal sequences (usually 5′) which have not been copied from the mRNA. This puzzles me a lot, but I have not been able to find ...
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Spectroscopic methods for quantifying peptides/proteins with or without Tryptophan or Tyrosine content
I have several peptides (20-50 amino acids long) which I want to quantify the solubility/concentration in a solvent at certain temperature and pH.
These peptides may or may not contain Tryptophan or ...
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Lab device to periodically, automatically transfer zooplankton between containers
I need a device to periodically draw a sample of transfer small aquatic organisms from one container to another without crushing/killing them. Each sample would be on the order of 10ml.
My sketch of ...
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What is the best way to stack parafilm wrapped petri dishes?
I have several petri dishes that are individually wrapped with bemis parafilm. As space is somewhat limited, I've begun stacking them on top of each other, however in doing this the wrapping for each ...
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Has anyone who has ever isolated synaptosomes using subcellular fractionation before know what the 'crude/heavy membrane fraction P2' is?
I am reading a journal paper where they analyse the proteome of synaptosomes. In this paper, they isolate synaptosomes from the hippocampi of mice. I know that synaptosomes contain the complete ...
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How long is agar agar good for?
I'm a high school science teacher, and am starting at a new school. I'd like to do some labs with bacteria and so I'll need to make plates with agar agar. I found 3 huge jugs of powdered agar in the ...
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How should I measure the oxygen dispersed during photosynthesis in pondweeds?
I am to conduct a lab investigating how different wavelengths of light affects photosynthesis in Egeria pondweeds.
The idea is to put color filters on light bulbs and shine them on the pondweed in a ...
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Transformation and PCR in molecular cloning
After obtaining the recombinant DNA, it is common to transform into E. coli to screen for recombinant DNA and amplify it. But I would like to ask can we amplify it using PCR?
I think there will be 3 ...
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What is the difference between 4th generation sequencing and NGS?
The generation of sequencing technologies has come on leaps and bounds and there are stark differences between the types of technology used. There is a great Q&A here What is the difference ...
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What antibody targets are being tested for in the publicly offered UK antibody test?
In late August 2021 the NHS (UK) offered people who test positive for COVID what is referred to in this BBC report as a “new antibody test”.
However, I have been unable to find out what exactly is new ...
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Why would this viral strain-specific antiserum fail to immunoprecipitate the same (98% identical protein) from another strain?
I'm reading this paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC392475/ and I can't work out why a certain immune serum didn't work on the same viral protein but from different strains.
The serum ...
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How do I cover Cellstrainers?
I‘m doing some experiments for my bachelors thesis and I‘m using some Cell strainers by Roth for it (https://www.carlroth.com/com/en/accessories/cell-strainer-easystrainer™/p/cly9.1). My problem is ...
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What's happening in the "C" and "T" stripes of a covid test kit?
I have a COVID home test kit which produces C and T (control and test) stripes when the solution is applied to the strip. Something similar happens in pregnancy test kits.
I understand the purpose of ...
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Sabouraud dextrose agar breaking?
I recently started growing some Geotrichum Candidum (GC) on Sabouraud dextrose agar (SDA) beds. The SDA was poured into sterilized petri dishes. After setting the plates in a cooler (20 C) for a day, ...
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Tips for longer fragment size and higher purity of insect DNA
Aim
In a pilot experiment I tested three different genomic DNA extraction methods on the non-model organism Pieris mannii (southern small white; Insecta, Lepidoptera, Pieridae). The goal was to ...
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Methods for getting clean C. difficile spore preps without spores clumping together
I'm a microbiologist recently who recently started working on an ongoing C. difficile project for the first time. One of the things I need to do is verify the lower detection limit of my qPCR ...
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Isolating colonies in a pure culture
I want to conduct antimicrobial susceptibility tests for which I've to culture bacteria first. Since I've bought KWIK-STIKs, (KWIK-STIK contains a single lyophilized microorganism which have to be ...
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dilutions in bacterial culture
In overnight bacterial culture, why do we put bacteria in a small flask to culture before moving it to a larger flask? Why not just put the sample in a large flask to begin with? This is in reference ...
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Washing buffers for protein identification?
I am currently learning protein identification techniques and come across ELISA and Western Blot. In these methods, a washing buffer is required to wash out the antibodies that are not bound to the ...
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Procedure of diagonal electrophoresis
I am learning protein sequencing and it seems that diagonal electrophoresis is a common method in the identification of disulphide bonds (S-S) exist between polypeptides in a protein.
Questions:
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How to identify plasma cells that only produce monoclonal antibodies?
I am studying the procedures of forming hybridoma cells for generating a large number of monoclonal antibodies.
Before the procedure of fusion (with multiple myeloma cells) happens, I would like to ...
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Which method of gene amplification for toehold switches?
My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience ...
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Is there a scale suitable for continuous tracking and recording of plant weight?
I want to measure water transpiration and evaporation in a bonsai tree, and measuring the weight of the tree in its pot is my proxy for water usage. This has worked well with manual measurements, but ...
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Can GFP reporting be used to track localization of peptides in the ER, Golgi, and plasma membrane?
Suppose I want to study the trafficking of a peptide throughout the ER, Golgi, and plasma membrane. An idea I had was labeling a secreted or plasma membrane integral protein with GFP and using time-...
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How can I clone a gene into a plasmid vector with an N-terminal his tag and TEV cleavage site between the tag and the start of the sequence?
I'm a scientist who has significant experience in chemistry but am relatively new to molecular biology and biochemical techniques. I'm trying to make an isolated domain of a protein (166 residues, 19....
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Enzyme inhibitor leads to higher turnover rate?
I'm currently working on a project where I have to deal with enzyme inhibition.
The purified enzyme shows a good substrate turnover. When I try to inhibit it with different inhibitors described in ...
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Alternative dyes for Gram staining
In the Gram stain, is there any replacement for primary stain, secondary stain, decolourizer and mordant? What results will the replacements produce?
I found that crystal violet can be replaced with ...
user64858
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How pricey does the media need to be for a typical bacterial culture experiment?
I'm considering various lab supplies for a protocol we're going to be running, and have been struck by the remarkable difference in price for different quality levels of the same basic substance.
...
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Saturated Mutagenesis Screening
I am hoping to mutate the active site of the enzyme I am researching that has 5 residues in proximity with the substrate. I am wondering how many colonies I'll have to assess to theoretically sample ...
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Assembling small DNA parts using Golden Gate
Background
I've always been told that DNA assembly can be tricky when using very small DNA parts due to low efficiency. I've also seen this when using 3A biobrick assembly to assemble promoters and ...
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How standardized is lab-grade skim milk?
I was surprised to learn in the answers to this question that in many cases the preferred cryoprotectant for many organisms is skim milk, rather than something more simple and well-defined like ...
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Measuring luminescence in a fluorescence plate reader
We're considering organizing some interlaboratory work on calibrating luminescence reporters (e.g., luciferase), and one of the key questions I don't know the answer to is whether most plate readers ...
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How long will a typical bacterial strain keep in a -80°C freezer?
I know that a -80°C freezer is the recommended means of long-term cell-line storage, and that cells will typically not last long in a -20°C freezer. But how long will a typical bacterial strain (e.g.,...
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