Questions tagged [lab-techniques]

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

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24 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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56 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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46 views

What is special about Pulse-Amplitude-Modulation (PAM) fluorometry?

I know that PAM fluorometry is used to measure chlorophyll fluorescence and understand the principle of those measurements. It is in principle clear to me from prior research in the literature, that ...
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1answer
84 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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1answer
110 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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3answers
83 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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3answers
162 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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149 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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1answer
45 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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4answers
283 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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2answers
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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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1answer
32 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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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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1answer
36 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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1answer
46 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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44 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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1answer
28 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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12 views

Why is transferrin saturation so low?

Transferrin Saturation (TS) is a common laboratory test and its values can be used to diagnose a variety of medical conditions. In healthy humans, and I assume it is the same for other species, TS is ...
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36 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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1answer
18 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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20 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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1answer
78 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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1answer
37 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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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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question on brain dissection

I was dissecting a sheep's brain for my students and one of them asked a question about the corpora quadrigemina he asked why did two of them(the superior colliculus) look more red than the other two ...
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55 views

Does glycerol in E.coli culture media somehow inhibit the lac-operon?

I have have been taught that one should induce protein expression with IPTG at an OD of about 1.0 - 2.0 when E.coli grows it TB media (terrific broth). As a reference point, one typically induces ...
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1answer
45 views

bunsen vs meker for best sterile field [closed]

I need one bunsen and i have a friend that sells me a bunsen-meker burner. I see that bunsen-meker burners have more flame diameter, so should have more diameter of sterile field, but i dont know XD ...
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1answer
145 views

1% water solution of deoxycholic acid. How is it prepared? [closed]

According to Sigma, the solubility of deoxycholic acid is: 0.24 g/L in water at 15C According to the FDA, Kybella (trademark) is a 1% water solution of deoxycholic acid. How did they do this?
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39 views

Why is my DNA band bulging?

This is the only image my the TA was able to get for us. And, we're using it for our lab report. The image isn't even ours. It's another group's image that we're sharing. But I don't understand why in ...
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1answer
32 views

Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
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1answer
19 views

Low-tech or low-cost technique for quantitative estimation in enzymology

If an accurate measurement of enzymologic quantities is needed, then following established methods in the field is necessary. However, it is sometime of great usefulness to ballpark a value before ...
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3answers
92 views

Book recommendation: protocols and recipes handbook for molecular biology / biochemistry

I'm a 5th-year PhD student in chemical biology. I've mostly been doing computational work, so my bench skills are rusty. To help me, I'd like a handbook of common techniques -- transformations, ELISA, ...
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50 views

Coronavirus and temperatures

An article on bioRxiv, Evaluation of heating and chemical protocols for inactivating SARS-CoV-2, recommends certain treatments to inactivate SARS-Cov-2 for lab work. The abstract notes: "Although ...
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3answers
2k views

Why does an autoclave need to pressurize steam?

I've looked this up on other sites and they say that an autoclave needs high pressure in order to raise the boiling point of water so that you can achieve steam of a higher temperature. If you look ...
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26 views

Can I use a Flask Weight Ring to increase RPM in an orbital shaking incubator?

I am working on a device that goes on top of the flask. This reduces the maximum RPM that I can use. Therefore I am wondering if I can use a Flask Weight Ring for Erlemeyer flasks in a orbital ...
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1answer
20 views

How is in-vitro fertilization technically achieved?

I failed searching on Google technical details about how in-vitro fertilization is achieved. I'm referring to the toolset and its use, rather than the chemical or biological aspects of the process. I ...
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1answer
34 views

How does the choice of blood draw site influence the possible specificity of a serological test?

The news has reported that a new serological test for the presence of anti-SARS-CoV-2 antibodies has received an emergency use authorization from the FDA, and notably has a higher specificity than ...
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36 views

qPCR to detect viral load

In an experiment qPCR was used to determine viral load. Alongside this conventional PCR was also performed and ran on an agarose gel. However I do not understand why both methods would have been used, ...
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1answer
130 views

How does SDS-PAGE separate based on mass?

In SDS-PAGE, electric field and mass-to-charge ratio are approximated to be constant for all proteins. Also, if $F=qE =ma$, then $\frac{m}{q}a=E$. Thus, all proteins must migrate with a constant ...
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15 views

How to choose an assay for detecting virulence factors?

When looking for a protein of interest, an ELISA is being administered. I read this article about the different types of ELISAs and the advantages/disadvantages. However, I am conflicted about ...
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2answers
73 views

Using autoclaved store-bought distilled water for labwork?

I'm a high school student who's working on a molecular biology project in my school's lab. I need pure water for making culture media, buffers, running a BCA protein assay, running digests, cleaning ...
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40 views

RNA product storage in isopropyl alcohol

I have an issue. I isolated RNA from cortex a few days ago. For isolation of RNA i used this procedure: I homogenizised my sample (mouse cortex) with Trizol. Then to the sample add chloroform, shooke ...
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1answer
19 views

Minimum amount of RNA for cDNA conversion and qPCR

I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased ...
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29 views

What is the most durable electronic pipette?

We have a routine task of pipetting substance concentration series in triplicate on cell cultures under a laminar hood. To speed up the process, we use an electronic pipette in the repetitive ...
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18 views

Are controls needed for urine cultures?

If testing for bacteria (gram-negative) in a urine culture, are negative or positive cultures needed? I could not find anything that says controls are needed unless results seem to be contaminated. ...
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1answer
123 views

Lab technique to distinguish between single stranded and double stranded DNA?

What lab techniques exist to differentiate between single-strand and double-stranded DNA?
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1answer
95 views

Why spin live fruit flies in centrifuge?

I was reading this article about the evolution of monarch butterflies’ resistance to cardiac glycosides: These Butterflies Evolved to Eat Poison. How Could That Have Happened? And this passage made me ...
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58 views

Bacterial Growth Formula

What are the units of $N$: the colonies of bacteria or the viable cell count? This is in regards to the bacterial growth formula used during serial dilutions, $$ N =N(0) e^{kt} \quad . $$
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67 views

Autoclaving media Question

1.When I autoclave media should I close the cap tightly or slightly? I put all tools in plastic bag when I autoclave, should I close slightly the plastic and when it finish I close tightly the ...
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1answer
380 views

Why is sorbitol used in buffers?

Many protocols in my lab use sorbitol in buffers. For instance, in co-immunoprecipitation, we include it at a final concentration of 200 mM in our lysis buffer. I'm not entirely sure why though. I ...

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