Questions tagged [lab-techniques]

Questions relating to protocols, procedures, and good practice when using laboratory equipment.

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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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2 votes
0 answers
23 views

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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yeast culture live cell extraction

I have been extracting samples for a gene expression assay from yeast. I would like to know how to ensure that the samples I am collecting at 48 hr. culture time, by then culture is probably saturated....
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3 votes
0 answers
25 views

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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2 votes
1 answer
78 views

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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4 votes
1 answer
100 views

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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How do refractive index (RI) matching solutions reverse tissue expansion in CLARITY tissue clearing?

I am learning about the CLARITY tissue clearing technique. I am researching this technique to understand how it works better. I know that this technique can make tissues transparent without severe ...
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3 votes
0 answers
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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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0 votes
1 answer
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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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1 vote
1 answer
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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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0 votes
2 answers
91 views

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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3 votes
1 answer
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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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1 vote
1 answer
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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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1 vote
1 answer
192 views

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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-2 votes
1 answer
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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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4 votes
1 answer
126 views

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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1 vote
0 answers
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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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10 votes
1 answer
133 views

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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1 vote
1 answer
59 views

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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9 votes
2 answers
460 views

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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5 votes
0 answers
38 views

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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3 votes
1 answer
67 views

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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5 votes
0 answers
39 views

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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2 votes
1 answer
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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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  • 343
2 votes
1 answer
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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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1 vote
1 answer
56 views

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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6 votes
2 answers
87 views

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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0 votes
1 answer
43 views

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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1 vote
1 answer
61 views

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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0 votes
0 answers
81 views

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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0 votes
1 answer
117 views

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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4 votes
1 answer
361 views

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 ...
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2 votes
3 answers
105 views

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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5 votes
3 answers
184 views

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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9 votes
3 answers
176 views

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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5 votes
1 answer
54 views

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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7 votes
4 answers
375 views

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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15 votes
2 answers
3k views

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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  • 6,937
2 votes
1 answer
252 views

What does it mean to "cure" microscope slides?

I am reading a journal paper and I have come across the following statement in the materials and methods section regarding the preparation of microscope slides: Coverslips were washed once more with ...
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  • 1,561
21 votes
4 answers
2k views

Pipetting: do human experimenters need liquid class information?

I've been working on a protocol standardization project where, among other things, we want protocols to be able to be run equivalently by both humans and robots. Something that I've noticed in doing ...
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  • 6,937
0 votes
1 answer
99 views

What is meant by 4 –12% or 8% SDS-PAGE?

I am reading a journal paper and I am looking at the materials and methods section. Regarding the Western blot method in the paper, I have come across the following statement: Proteins were separated ...
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2 votes
1 answer
87 views

Why when measuring turbidity do we use the minimum wavelength?

As a preface, I read a few other related posts and was able to gather some knowledge, though without any background in physics I am having some trouble here piecing together a coherent view. I looked ...
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0 votes
1 answer
91 views

Is there any reason to use an upright microscope over an inverted microscope?

I've never actually owned an inverted microscope, but it seems it has only advantages compared to an upright microscope: taller, heavier samples; no crashing the objective into the glass slide; easier ...
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1 vote
1 answer
33 views

Reliable recipe to make DIY yeast slants

Does anyone have any good ideas on a reliable way to activate basic grocery store yeast? I realize this is a total beginner's question. I've built a small yeast ranch at home with my kid, and we're ...
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0 votes
0 answers
58 views

What statistical test would you recommend to compare two animal behavior sampling methods?

I am doing a lab where I am comparing the scanning method and focal animal method. Each method will give me the following: Scanning method: occurrence frequency for each behavior (%), focal animal ...
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1 vote
1 answer
44 views

When should I use cryofixation and chemical fixation?

We know that the technique used in TEM sample preparation involves multiple steps, one of the most important of them is fixation. Fixation can be of two types: Cryofixation, that suggests that the ...
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0 votes
0 answers
23 views

How to do manual Saccharomyces tetrad separation with minimal equipment?

I would like to separate the four ascospores from individual asci by manual microscopic manipulation without at micro-manipulator and a basic dissecting microscope capable of 600X total magnification ...
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2 votes
1 answer
92 views

Decreasing signals in assay measurements

I'm working with a calcium assay to study the effects of different virulence factors. The assay works, but from day to day the signals of cell lysis go down. Unfortunately, I haven't found an ...
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2 votes
1 answer
54 views

How to attract Cyanobacteria?

I would like to attract cyanobacteria in one spot on an object (e.g. cloth,) instead of having it swim in the media. my current method is to pour it on the object in a beaker and wait for some of them ...
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12 votes
4 answers
2k views

Are bleach solutions still routinely used in biochemistry laboratories to rid surfaces of bacteria, viruses, certain enzymes, and nucleic acids?

Decades ago I would stop by biology and biochemistry laboratories to see what was up, and I noticed a ubiquitous presence of a squirt bottle of bleach solution. When I asked what it was for I would ...
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