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Questions tagged [lab-techniques]

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

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188 views

Should I use RNAseZAP on my bench and pipettes before RNA extraction?

Should I spray RNAseZAP on my bench and pipettes before working with RNA? I am regularly using RNase at my bench and would like to work with RNA as well. Could traces of RNase on my equipment degrade ...
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23 views

How to convert a test tube assay to microplate assay?

I am trying to measure Ascorbic acid levels in tissue using Omaye et al protocol (Methods in Enzymology, Volume 62, 1979, Pages 3-11). This method is based on reduction of ferric chloride and ...
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30 views

What is known and what can be measured of biofilms?

Context for the question I am a mathematics researcher working on the mathematics of imaging and parameter estimation (inverse problems). I found an interesting diffusion equation that models the ...
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1answer
127 views

DNA quantification in a high school bio lab

I'm working on a project in a high school bio lab (so limited resources), and I need a way to quantify the concentration of DNA in a PCR product. I can't use spectrophotometry (cheap ...
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1answer
36 views

Why marker and control are loaded in the beginning or ending lane of the gel?

I have seen gel images in several literature. Almost in all, markers and controls are loaded in the extreme lanes (before 1st sample or after last sample) of the gel. I am just curious, is there any ...
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2answers
1k views

Is working with (nitrile) gloves around a bunsen burner safe?

Recently I have started in a new microbiology lab, and with a new lab come new habits. When I was working with my bacterial (liquid) culture next to the flame (Bunsen burner) wearing nitrile gloves, ...
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44 views

Can anyone expolain this? Cell culture vials have strange hair-like condensation patterns

What is the hair-like polymer that condenses on the caps of the plastic cell vial? Inside vial are cells in a mixture of FBS and DMSO. The vials were frozen slowly in a special isopropanol-filled ...
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1answer
32 views

HELP hepes preparation [closed]

I need help: I would like to prepare an HEPES solution, without using the powder. It is possible? I remember that I did it once, but maybe I'm wrong...does someone know a protocol for HEPES ...
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1answer
140 views

Optimization of Signal Peptides

We are looking at modifying the signal peptide of a receptor in a common immortalized human cell line. The cell line already expresses an unusually high amount of the protein, but much of it is not ...
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0answers
160 views

How to keep Paraffin Sections from falling apart?

I've been trying to make 5 uM sections of injured mouse spinal cords and stain the tissue with Luxol Fast Blue and H&E. At first I was removing the bones from around the spinal cord, but the ...
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2answers
767 views

Agarose gel Ladder smear

We are having trouble with agarose gel electrophoresis. It used to work a couple of months ago but now the ladder always look smeared. We switched the components (1x TBE, 100bp ladder, different type ...
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0answers
82 views

What happens during fixation of Nucleic acids on nylon membrane

In Southern blotting technique, fixation of DNA to nylon membrane is a step prior to hybridisation with probe. It is done by baking at 80ºC and according to this cross linking between DNA and nylon ...
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1answer
2k views

What is the role of glucose in plasmid isolation?

My understanding is that glucose is used in the resuspension solution to prevent cells from bursting by maintaining the appropriate osmotic pressure. Why do we even bother doing this? The cells are ...
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1answer
204 views

How can the melting temperature of PCR primers be so far below the extension temperature?

Since our primer TM is lower than the extension temperature by design, (and often by a significant amount) shouldn't the primers be released from the template at extension temperatures? Now, I have ...
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2answers
915 views

Why does high EDTA concentration cause swelling of platelets?

According to this textbook, high ( >2mg/ml of blood) EDTA concentration causes swelling of platelets which lead to their fragmentation. I know that high concentration of EDTA increases plasma ...
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0answers
37 views

How urination prior to blood test may give error?

Source: Textbook of Practical Physiology, 4th Edition according to it urination within 30 minutes is a precollection factor that may alter the results of blood test. I am unable to comprehend how ...
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1answer
77 views

How to watch a Zebrafish embryo in detail?

How much microscope zoom would I need to watch the development of a Zebrafish embryo in certain detail? Thanks guys!
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0answers
13 views

Propagating errors for percentage reduction

Need a bit of help for this particular problem. I have three averages collected from three genotypes (A, B, and C) and their associated SD (a, b, and c). A is my control. I have been asked to ...
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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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90 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 ...
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1answer
159 views

Help with preparing Congo Red TSA

So I want to make Congo Red TSA. My instructor gave some hints but I still have some questions: I start by making two stock solutions: 20 mg/ml of congo red and coomassie in two different falcon ...
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1answer
121 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 ...
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63 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 ...
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0answers
53 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 ...
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2answers
869 views

Lab marker for labelling cell culture plastics?

I am a lab manager trying to ask other lab biologists what brand/make of markers their labs use for labeling tissue culture plastics, which need to be repeatedly wiped down with 70% ethanol to ...
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1answer
624 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 ...
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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 ...
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308 views

Why is thrombin time (TT) normal range longer than prothrombin time (PT)?

Reference range for the TT is longer than that of the PT.
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1answer
100 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 ...
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1answer
590 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 ...
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207 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 ...
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1answer
154 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, ...
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1answer
799 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. ...
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1answer
133 views

How do I improve expression using the GAL1 promoter in S. cerevisiae above the “leaky” level I'm currently seeing?

I am attempting to express GFP in S. cerevisiae using the GAL1 promoter. I always grow an uninduced (in dextrose based SD medium) culture alongside my induced (in galactose based SD medium) culture. ...
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1answer
216 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?
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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 ...
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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?
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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 ...
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1answer
448 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. ...
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2answers
710 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 ...
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1answer
2k 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 ...
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1answer
1k 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 ...
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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?
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1answer
740 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 (...
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1answer
139 views

Method for preparation of LB agar plates with Ag+?

I need a method for preparing LB agar plates with 1 mM concentration of AgNO3. I can not seem to find a method online, does anyone know of one that I could use? Here are my main questions regarding ...
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1answer
202 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; ...
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1answer
281 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 ...
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62 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 ...
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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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86 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 ...