Questions tagged [lab-techniques]

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

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1answer
146 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
249 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
5k 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
508 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. ...
3
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2answers
864 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
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 ...
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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?
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1answer
811 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
146 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
216 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
350 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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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 ...
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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
92 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 ...
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1answer
176 views

EnteroPluri test contamination

An EnteroPluri test has twelve different sectors. It seems odd to me that pulling the inoculating tip from the starting sector (citrate) all the way to the ending sector (glucose/gas) and then ...
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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 ...
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1answer
294 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 ...
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1answer
5k 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 ...
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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.
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1answer
843 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
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 ...
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1answer
231 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 ...
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0answers
285 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....
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1answer
443 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?
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2answers
81 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
892 views

Collecting virgin fruit flies [closed]

Suppose that you place P generation wild type males and mutant virgin females in a vial and allow them to mate. You leave this vial undisturbed in the incubator for 12 days. Could you collect F1 flies ...
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2answers
88 views

Common mistakes when sequencing?

I've sequenced two genes of 15 dogs for SNP analysis. the chromatograms of one gene looked perfect but the ones of the second gene didn't look as good. To be precise, the chromatograms of one exon ...
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0answers
49 views

What is the typical amount of full length clones in a cDNA library?

For a Yeast Two Hybrid project we need to make a cDNA library from a single celled organism. We used the Cloneminer II kit from Thermo. We followed the manual and performed all the quality control ...
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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 ...
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0answers
68 views

PCR limits of detection calculate method

Is any standard method for use to gain PCR sensivity or limit of detection? my mean is not for RT-PCR or Real-Time or any other kind and just simple PCR. After making PCR product free of dNTP and ...
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0answers
76 views

Do users of CRISPR/Cas iterate or parallelize to try multiple guide sequences?

I've read about on-target efficiency and off-target effects in use of CRISPR/Cas9, and about tools that suggest good guide sequences. I am wondering: how many guide sequences do typical CRISPR users ...
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2answers
432 views

What is the best way to analyze non-quantitative mass spec hits from an immunoprecipitation pull down?

I am studying a nuclear protein and want to come up with a list of potential proteins that it interacts with. From the nuclear fraction of 293T cells, I did an IP (immunoprecipitation) to pull down my ...
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1answer
2k 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
2k views

PBST vs. TBST buffer in western blotting

What is the advantages and disadvantages of using either PBST or TBST in western blotting, or while working with proteins in general? Are there other buffers which are also used for western blotting, ...
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1answer
476 views

Optimal pH of protein buffer? Basic principles to adjust buffers according method and analysis

Protein buffers such as PBST, which is used in western blotting, are normally adjusted to pH 7.4. When I try to find why, I find some information about optimal pKa for protein stability. Im not sure I ...
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1answer
1k views

Can RNA be extracted from tissue suspended in formalin?

There are two tumor samples floating in a 10% formaldehyde solution (i.e formalin). Is there a protocol for RNA extraction under these circumstances? I am concerned that using the protocol for ...
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1answer
715 views

Common Errors For Low R Value in Bradford Assay

I've recently started doing Bradford Assays for my samples and my standard curve has been non-linear and I have been getting low R values (.90-.95). I initially thought the error was in pipetting, ...
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1answer
2k views

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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0answers
265 views

How is the acetone method different from QB buffer for extraction of plant protein?

I primarily work on animal tissue but I thought of running an SDS-PAGE for plant tissue just for fun. I searched for some protein extraction protocols online and I don't know which one to choose. One ...
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1answer
99 views

How well do Eppendorf cups seal?

I am considering to send some samples overseas in Eppendorf cups. They are the standard plastic cups of 1 to 2 mL capacity. Of course they may tip over during their journey and I'd like to know how ...
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0answers
13 views

Hydrophobic elution times in ionic exchange columns

What would the order of elution be of say Gly, Val, Leu? My argument is that hydrophobic residues will try and get away from the resin and the more hydrophobic the residue is the faster it will elute....
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1answer
44 views

Transfer of electrophoresis bands to MS

I have a lab question that is confusing me. Just to be clear this is a homework question but I've done some extensive research and can't find what seems to be a "good" answer. The question is this: ...
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1answer
432 views

How does 2-mercaptoethanol lead to shift of the band to a higher molecular weight?

I have a project to isolate a protein with biological properties from a plant. The purified protein forms four bands with similar molecular weight on SDS-PAGE (30–35 kDa) in the presence of 5 % 2-...
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2answers
4k views

What is the meaning of a reported OD600 value?

I can't find a reference that explains what, for example, OD600 = 0.1 means. For example, Wikipedia states OD600 is an abbreviation indicating the absorbance, or optical density, of a sample ...
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2answers
714 views

Does denaturing proteins lead to loss of epitopes?

I am doing an experiment where I have to do both Immunohistochemistry and SDS-PAGE. I am assuming that the native conformation of the protein is maintained in the IHC. But during the blot, we heat the ...
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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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1answer
110 views

Minimal viable EEG equipment for dissertation research on BCI / BMI

I am planning out a dissertation study of Brain-computer Interface (a.k.a. Brain-machine Interface, BCI, BMI, etc) applications. One of the 3 papers in that dissertation will involve collecting ...