Questions tagged [lab-techniques]

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

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7
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2answers
1k views

Alternatives to CFU plating for measuring number of viable cells?

I am hoping to measure growth rates of a bacterial culture in several growth conditions. I am concerned that these growth conditions may cause cell death, which would lead to a decreased ...
2
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0answers
35 views

Nucleic acid extractions

Is it fine to leave pulverized plant tissue in CTAB buffer at -80 degrees over night? will the buffer remain effective to generate good quality nucleic acids?
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0answers
97 views

What are preparation mistakes that could make red blood cells appear crenate in isotonic solution?

Thank you for your time. I separated red blood cells from a whole blood sample purchased from a blood bank. In isotonic solution, the red cells appear crenate. I would like to know if there is ...
1
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1answer
128 views

Is it possible to produce a tangible mass of DNA/RNA using a PCR reaction?

A Polymerase Chain Reaction (PCR) is a method of amplification which enables one to produce many copies of a certain DNA/RNA strand for many applications (as described in the link above). I have ...
7
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1answer
4k views

Role of calcium chloride during competent cell preparation

I am aware of the fact that $CaCl_2$ settles down on the cell wall making it less negative may be by forming bond with Teichoic acid. Also due to the positive charge it attracts DNA (DNA is negatively ...
2
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1answer
820 views

microscopic slide cleaning and maintenance

we do lot of gram staining and some spore staining. we normally wash our slides with detergent then rinse in D/w, dry it and store it. But on reusing this slide we have to flame it and also clean it ...
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0answers
387 views

Recommend any Molecular lab LIMS (Laboratory Information Management System)

We are looking to develop or customize a LIMS for our molecular lab. Do you know of a LIMS that you've used in a molecular lab before, or one that could be used. Thanks for the help (Edit) Some of ...
4
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4answers
285 views

Electronic laboratory notebook (ELN)

I have been using old style lab book for some time now but with increasing work on computer and storing sequencing results and gel pictures on computer it would be nice to have everything on computer ...
1
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3answers
2k views

Why do people remove excess solution after they put on a coverslip?

Today, my classmate and I stained a slide of cheek cells. After we put on a coverslip, our teacher said that we are supposed to remove excess solution using a tissue. Why should we do this? I am so ...
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0answers
75 views

Plasmid transformation yields only empty vectors

I have to electroporate pMAD cloned with an insert of 7.4 kb in my S. aureus isolate. When I electroporated in RN4220 I had no problems but when I put it in my isolate I just recover few colonies and ...
6
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1answer
12k views

What are the roles of guanidine-HCl and ethanol in binding of DNA to silica?

I'm trying to understand how exactly the binding to silica gel (in kits) step works and I cannot find any papers which provide an explanation of the physics or chemistry; especially on the way that ...
3
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1answer
105 views

correction of read counts for spikes

I have 4 RNA-seq samples (1st is control and the other 3 are treatments) and 5 spikes. I used different concentrations for 5 spikes but over different samples, I used similar concentration. for ...
2
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1answer
837 views

Effect of ethanol and detergent on cell membranes problem

I am currently writing my biology report for an experiment I did on how ethanol and detergent affect the cell membrane. For my ethanol experiment, all went as expected. However, for my detergent ...
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0answers
29 views

What is the proper way to dispose of a medicinal substance?

Is there a protocol or a set of guidelines to safely dispose off a batch of expired drugs (as a manufacturer)? I guess the process may vary for different drugs. My specific question regarding the safe ...
1
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0answers
34 views

How could you measure the rate of light reactivity/ photosynthesis of a cell in a lab setting?

For a lab I must take Volvox protist cells and expose them to specific wavelengths of light. How would I know they're even reacting? The assignment I've been given does not explain what I should even ...
4
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2answers
501 views

Ideal lab glassware cleaner for molecular biologist

I'm using Alconox powder detergent to clean my laboratory glassware, the powder is a pain to store near the sink and to use. Would like to make a concentrated liquid to use in its stead. I have ...
4
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3answers
5k views

DpnI over-digestion

We have a long protocol that we are optimizing that includes DpnI digestion of a PCR product (to remove any of the template DNA if it's methylated, and while we're not certain in the blind tests, ...
7
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1answer
2k views

Alfred Jost's rabbit experiment: how did he actually do it?

I was watching a Crash Course on Biology YouTube video by Hank Green: Great Glands - Your Endocrine System. Hank humorously describes a sex determination experiment by Alfred Jost as follows (...
2
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0answers
54 views

plausible science for why an enzyme breaks down a substrate better at 4 degrees rather than 20 degrees [closed]

An enzyme was found to have an optimal temperature of 20 degrees. The same enzyme in sheep liver extract was found to have an optimal temperature of 4 degrees. What are some of the factors that can ...
1
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2answers
985 views

What kind of detergent should I use in cell culture laboratory for cleaning purposes?

I work on establish a cell culture laboratory in the company where I work. All equipment are new and I want to clean lab before I start to work with cells. Is there any special detergent for cleaning ...
5
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1answer
757 views

Are vacuum centrifuges the same as normal centrifuges?

I came across a method that used a vacuum centrifuge: Carvalho et al. 2012. The diversity, antimicrobial and anticancer activity of endophytic fungi associated with the medicinal plant ...
4
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1answer
670 views

Problems with SDS-PAGE, no separation - what could be the problem?

I work in a lab were we have a common stock of 30% Acrylamide, TEMED, TRIS-HCl (pH6.8 and pH 8.8.) and a 10% (w/v) SDS solution pH 7.2. We use the recipe for loading buffer (which we of course mix ...
17
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2answers
16k views

What effect does vortexing have on a fluid sample that simple mechanical shaking does not?

Some protocols call for fluid samples to be mixed with a "vortexer" on the high setting. What effect does the vortexing have on fluid samples that mechanical shaking does not? Does it shear long ...
3
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1answer
311 views

How long does Lentivirus take to express in vivo mouse neurons?

Does anyone know how long it takes for a standard Lentivirus vector to express its genes (under a strong promoter such as CAG, CB7, etc.), after injection into the brain of a mouse? By hearsay I ...
4
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1answer
3k views

How to convert skin temperature to core temperature?

As opposed to regular invasive thermometers, there are non-invasive IR thermometers to measure the temperature. For example Thermofocus. I read in many sources that these IR thermometers only measure ...
3
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2answers
841 views

When is strict sterile technique necessary? Cloning vs. protein expression

Background: I have previously worked with RNA, and then we use laminar flow hoods for any work where we do plating or inoculation of cultures while applying sterile technique. I am now working in a ...
5
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1answer
4k views

Why calf thymus DNA is widely used instead any other body part?

Calf-thymus DNA is widely used as DNA sample. Such as testing anti-dsDNA antibody activity, nuclease activity etc, as well as certain books include calf-thymus-DNA in various examples (such as the ...
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0answers
211 views

Why are my assay absorbance values very low?

I am culturing SH-SY5Y cells in 96-well plates at a seeding density of $1.2 \times 10^5$ cells/mL. I differentiate the cells with retinoic acid for 6 days before giving them treatments. The cells look ...
3
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1answer
160 views

FPLC based separation of serum proteins

I am working on a project which involves bio-marker discovery in neuro-developmental disorders. I have an akta-prime FPLC instrument in my lab but I do not know how to use it. According to theory, ...
5
votes
1answer
15k views

How to prepare sample for SDS PAGE?

I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
0
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1answer
878 views

Aseptic technique: a single technique, or any technique fulfilling criteria, or collection of said techniques?

My friend just used the following sentence in her lab report: Aseptic technique is used in this step: I immediately felt something was wrong. She used the term as if it were a specific technique. ...
5
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2answers
1k views

High Current (Speed) Transfer Buffer Recipe

Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
2
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1answer
246 views

During the process of plasmid isolation, how can we be sure that only plasmid DNA has been isolated and there is no chromosomal DNA?

We separate the membrane and all other cell components, but how is it that only plasmid DNA is isolated and not chromosomal DNA?
2
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1answer
2k views

Sterilize/disinfect sugar for lab use

How do I sterilize/disinfect ordinary table sugar for lab use? I'm using the sugar in an agar and I want it to be as clean as possible. Are there any effective, conventional ways of doing this?
1
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1answer
88 views

Fine permanent fluorescent marker for blue light

I am looking for a fine- (or small-)tipped permanent marker to write on Petri dishes that is visible under a blue light or a transilluminator. It would seem obvious, but it is a nuisance. The ...
3
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4answers
4k views

Measuring protein concentration, Bradford vs. Nanodrop?

I know that the bradford assay is a very standard way of measuring protein concentration after e.g. a purification. However, in the lab that I work in now they normally only use nano drop at the 280nm ...
3
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1answer
500 views

Protein purification: Linear elutions or stepwise elution, using imidazole or pH gradient?

I now work in a lab with a protein and a pre-made purification protocol, were they purify a His-tagged protein using linear elution which then gives a steady increase in the imidazole concentration. ...
1
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1answer
3k views

What type of immersion oil should I buy?

I have an old Nikon Alphaphot microscope that came with a 100x oil immersion objective, but no manual. I've seen plenty of sources that explain that you need to place a drop of oil between the slide ...
1
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1answer
2k views

Beginner question about growing E. coli

I have bought a hobbyist kit which involves growing E. coli. The steps said to grow the E. coli on an LB agar Petri dish overnight. No incubation devices were included in the kit. I let the E. coli ...
3
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1answer
3k views

Why are red blood cells preferred to study the structure of plasma membrane?

If we wanted to study the structure of a plasma membrane, why are red blood cells a more attractive cell type to work with than other cell types such as liver cells or kidney cells?
7
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2answers
751 views

poor RNA quality from zebrafish embryos

Does anyone routinely do RNA isolation from zebrafish embryos? I have embryos from different stages but all below 24hpf. This is the protocol I follow: Take 10-20 embryos Wash once with milliQ water ...
2
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1answer
808 views

What bacteria results in a Gram +ve cocci and catalase +ve? What test comes next?

Results so far. We are trying to determine unknown microorganisms in intro to microbiology course. I first did gram stain and they were all cocci morphology, purple color and clumped together (...
5
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1answer
209 views

What kind of “size” does SDS-PAGE separate by?

What is the exact property of the protein that affects migration of bands in SDS-PAGE; is it length, volume, or mass? For example, will GGGGGGGGGG (glycine 10-mer; ...
1
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1answer
337 views

How to pipette accurate volumes from BSA solution without air bubbles?

I'm new to immunohistochemistry and there's one step I'm having problems with. Generally I can pipet volumes pretty accurately and avoid air bubbles, but: 1) The BSA solution that I made (with PBS ...
2
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0answers
64 views

Where am I going wrong in a Reactive Oxygen Species Assay done in N9 cells?

I have followed the modified protocol that was given to me by my senior (who was using for his RAW 264.7 cells - Reference article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308605/ ) for ...
4
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0answers
2k views

How much salt [NaCl] is too much in DNA precipitation?

In DNA extractions, how much is too much salt in a CTAB extraction buffer? Protocols hover around 2.5 molar; if you go over this (e.g. 25 molar), will you saturate your solution, and precipitate the ...
1
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1answer
100 views

Measuring optical density with/without re-suspending cells

When measuring absorbance using a plate reader, is it necessary (or better) to resuspend the culture if the plate has been sitting still for 1-2 days? While this could be a good physics question, I ...
0
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2answers
92 views

probe amplification in MLPA

I'm reading an article about MLPA (Multiplex ligation-dependent probe amplification) and I got stuck on this sentence: The advantage of splitting the probe into two parts is that only the ligated ...
3
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0answers
91 views

Travelling with tissue samples in RNAlater

I've been in Brazil collecting some samples (ants) and need to travel back to to the UK with - I've got their brains stored in RNAlater, which have been in the freezer at -4C for a bit less than a ...
1
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1answer
293 views

How is monoclonality or polyclonality determined?

I was reading up Kaposi sarcoma and Robbins Pathology says, ..many features suggest that KS is not a malignant tumor despite the ominous name ...spindle cells in many KS lesions are polyclonal or ...