Questions tagged [lab-techniques]

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

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How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes? Some background: I'm trying to ...
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1answer
99 views

How can I save bacillus strains on filter paper without an -80 degree freezer?

I want to save my bacillus strains but I don't have access to a -80 degree freezer. What are possible alternatives?
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1answer
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Can I heat Trizol?

I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause ...
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1answer
355 views

Is DNA green viewer carcinogenic?

I use DNA green viewer in Lab to see DNA and RNA bands. Some peers told me it is carcinogenic and is not safe. Is this correct? If yes, are there better choices to use in working with gel?
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1answer
178 views

Test of antibiotic A for sensitivity as surrogate of antibiotic B

I am reading the WHO Global Antimicrobial Resistance Surveillance System (GLASS) - Manual for Early Implementation. it mentioned some of the drug-bug combination that will be put under surveillance in ...
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1answer
802 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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1answer
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For people who work in microbiology labs, what software do you use to manage your strains?

It's just a small microbiology lab that currently records everything on paper, and there's quite few mutants as well. Is Excel commonly used for this sort of thing? Or is there a better software to ...
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1answer
700 views

What is a simple protocol for staining cells in suspension?

I am an engineering student studying how electric fields affect cells, specifically the phenomena of electroporation in living cells. I know that electroporation is widely used for introducing genes ...
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1answer
73 views

Effect of Ethanol on Brain Volume Measurements

I want to compare the brain sizes of two populations of fish. However, all the samples I have were fixed in 95% ethanol. As far as I know, 4% formalin is the normal fixative for soft tissues. Can I ...
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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 ...
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83 views

Procedure for doing western blot [closed]

I am writing a step by step guide for doing a western blot for a class. It is intended for any one with basic Biology lab skills. I am hoping people will review my draft and give feedback on how to ...
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Can I purify polyhydroxyalkanoates by heating the cells extensively?

Traditional methods of purifying polyhydroxyalkanoates (PHAs) and other bioplastics made by bacteria involve washing the cells with harsh chemicals or strong bases.I'm interested in maintaining the ...
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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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3answers
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Improving Gel Extraction yields

How can I improve my Gel Extraction yields. We use the standard protocol from Qiagen, gel extraction, dissolve in QG buffer at 42C and purify via anion exchange columns. However, with 500 ng we ...
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2answers
1k 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 ...
3
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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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1answer
4k views

“X” in stock solutions

We prepare buffer solutions with concentration in terms of x, for example 50x TAE buffer. How do we come up with ...
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1answer
623 views

How to measure quality and quantity of DNA?

I would like to mesure DNA. I quantify the concentration with Qubit fluorometer, but I would like to know also quality of DNA. I try BioAnalyzer (Agilent),but without success. Bioanalyzer measure DNA ...
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1answer
131 views

Immunoprecipitation compared to western blotting

Immunoprecipitation and western blotting are both used to locate a specific protein within a sample and to isolate it. In immunoprecipitation, a specific antibody and agarose beads ( or other ...
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3answers
353 views

Using nanodrop for analysing biological samples other than nucleotides

I am a 3rd timer postdoctoral fellow with some experience in molecular biology and biochemistry, but major skills in Zoology and Natural History. I am studying some natural extracts, and isolating ...
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2answers
1k 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 ...
3
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1answer
89 views

Decreasing Solubility as Solvent Moves Higher in Paper Chromatography

In paper chromatography, as the mobile phase (I used 1:9 acetone/petroleum ether) climbs higher and higher, will the solubility of the solutes decrease as the solvent moves higher? I speak mostly ...
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1answer
196 views

Changing time and rpm of centifuge

Inspired by this question, I want to ask a question about centrifugation. Suppose a protocol says : 10 min at 2500 rpm . Can we instead centifuge for 5 min at 5000 rpm or 20 min at 1250 rpm ?
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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
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Forgot to vortex antibody before staining

Ugh. Did an immunofluorescence experiment last weekend, forgot to vortex both my primary and my secondary antibody solutions. And my final result looks dimmer than it should be. Is it possible that ...
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3answers
1k views

LCMS/MS versus Western Blot

I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different ...
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1answer
8k views

Why does the pET- expression vector contain a LacI gene additionally to the one in the genome?

The pET plasmid is used for protein expression with T7 promotor in expression strains, such as E.coli BL21(DE3) It contains a lacI gene which codes for the lac repressor protein, a protein of ...
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2answers
74 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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1answer
128 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
4k 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
1k views

Why is it advised to avoid bubble formation during mixing?

I have been told not to vortex solution containing protein. The reason I was given is bubble formation. Here I am interested in the effect of bubble formation in general.
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1answer
585 views

Benefits of CLARITY?

What are the benefits of CLARITY over this technique that was published more than a year earlier? Of course the second technique needs a fancier microscope that is likely more expensive and requires ...
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1answer
305 views

How do different tissue culture matrices affect background in fluorescent microscopy?

In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always ...
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1answer
546 views

How is the gradient in density gradient centrifugation made?

From the literature I read, I came to understand the following. Please correct me if wrong. In CsCl gradient centrifugation, the gradient is achieved by centrifuging the CsCl solution at high rpm, ...
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1answer
3k views

What would happen if a cell is poked by a fine needle?

I had seen this question in an exam: A living cell has a protoplasm which is water based and demarcated by a lipid bilayer membrane. If a cell is pierced to 1/5th of its diameter with a very sharp ...
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1answer
393 views

How can I purify RNA after gel electrophoresis to remove residual acrylamide?

I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still ...
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1answer
550 views

PCR master mix contents

I am running PCR reactions with different sets of degenerate primers and I want to know what should go into the master mix My usual master mix: DEPC water PCR buffer (-Mg) 25mM MgCl2 10mM dNTPmix ...
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4answers
880 views

Bacterial cell lysis buffer used in proteomics procedures

What kinds of detergent-free bacterial lysis buffers exist? The proteins we're extracting will be later analyzed by LC-MS/MS, and we're looking for a lysis buffer that won't interfere with this ...
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1answer
73 views

Non-fatal / low-harm measurement of Drosophila traits

I am trying to generate some candidate traits to measure in a fitness assay of wildtype outbred (lab population) flies. The key trait I will measure is lifespan. I am looking for some additional ...
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1answer
2k views

Optogenetics - How do microbial opsins work?

I'm just introduced to the optogenetics method and am having some trouble grasping the genetics (of the optogenteics) part of things. So we have Retinal and Opsin that form Rhodopsin molecule that ...
3
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1answer
302 views

205 nm UV-Vis readings

Typically we determine the concentration of proteins using a 280 nm reading. However, it is reasonable to use 205 nm. I was curious about the effectiveness of this method.
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1answer
181 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, ...
3
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1answer
657 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
94 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-...
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1answer
201 views

How C. Elegans is used for siliencing genes

The experiment that is using C. Elegans to silence the Genes. I have a question about Why and how C. Elegans can use the DNA plasmid that is generated with the gene of interest in the bacteria by ...
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1answer
195 views

Well to well variation in thermal cycler fluorescence?

We have an old BioRad ICycler Thermal Cycler with MyIQ single color fluorescent detector. While it's meant for RT-PCR, we've been using it for melt curves and binding assays for different types of RNA ...
3
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1answer
2k views

Problem with bacterial transformation with electroporation

I have a problem with a bacterial transformation of a yeast gene that I can not solve. I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest ...
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1answer
336 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 ...
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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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How does a heat-ratio sap flow sensor work and how invasive is it?

The Gizmodo article Undead Tree Stump Is Being Kept Alive by Neighboring Trees says: Leuzinger and Bader stumbled upon the stump while out for a hike. The woody stub caught their eye because callus ...

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