Questions tagged [lab-techniques]

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

Filter by
Sorted by
Tagged with
1
vote
3answers
1k 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 ...
1
vote
0answers
66 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
votes
1answer
11k 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
votes
1answer
102 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
votes
1answer
772 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 ...
1
vote
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
vote
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
votes
2answers
492 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
votes
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
votes
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
votes
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
vote
2answers
909 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
votes
1answer
677 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
votes
1answer
622 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
votes
2answers
15k 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
votes
1answer
308 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
votes
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
votes
2answers
751 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
votes
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 ...
1
vote
0answers
209 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
votes
1answer
156 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
14k 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
votes
1answer
850 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
votes
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
votes
1answer
225 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
votes
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
vote
1answer
87 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
votes
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
votes
1answer
459 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
vote
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 ...
1
vote
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 ...
3
votes
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
votes
2answers
747 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
votes
1answer
778 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
votes
1answer
206 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
vote
1answer
302 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
votes
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
votes
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
vote
1answer
99 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
votes
2answers
91 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
votes
0answers
88 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
vote
1answer
272 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 ...
2
votes
1answer
4k views

Purpose of dilution streaking or streak purification

In dilution streaking a drop of liquid culture is collected with an inoculating loop and then streaked across the agar plate surface. At the end of the streaking the number of organisms clinging to ...
2
votes
1answer
19 views

What for a Kinase Assay? [closed]

I am wondering why to use a kinase assay, since we can extract the proteins from cells and then do a Western with the specific antibodies we want to use.
1
vote
0answers
37 views

How did researchers find out that it was only the maternal chromosome that underwent deletion in Angelman syndrome?

It is known that in Angelman syndrome, the chromosome 15 undergoes deletion in the maternal chromosome while in Prader-Willi it's in the paternal. I understand it can be detected by FISH or other ...
2
votes
1answer
1k views

Strange behavior of a DNA gel

I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sybr-safe as a DNA stain. Voltage was 80V. When I imaged the gel, the DNA on the bottom half of the gel, including the ...
1
vote
1answer
215 views

Measuring Bioluminescent Algae Intensity

For a science project, I am required to measure the intensity of the light given off by the Algae, Pyrocystis lunula. Can a photometer with a range of 1-50000 lux be able to pick it up? If not, what ...
3
votes
2answers
77 views

Technical term for water entering a semipermeable membrane?

Would the word happen to be diffuse? I have tried imbibtion but that word is invalid because it isn't specific to osmosis. Research: http://www.majordifferences.com/2013/12/difference-between-osmosis-...
-1
votes
1answer
410 views

How to incubate freeze dried culture?

I have a freeze dried culture of staphylococcus aureus; how do I incubate it? Can I just mix it into Luria broth and just keep it in the incubator?
1
vote
0answers
271 views

DAPI staining showing unknown artefacts?

Dear fellow researchers, I have been facing some problem with staining nucleus with DAPI. I use the standard protocol for fixation 4%PFA+0.1% TX-100+2%BSA. I use 0.2ug/ml conc. of DAPI for 5 minutes....