Questions tagged [lab-techniques]

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

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1answer
392 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
65 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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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 ...
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1answer
101 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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2answers
94 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 ...
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96 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
339 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
20 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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38 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
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 ...
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1answer
233 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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2answers
91 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
460 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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293 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
130 views

Can microdialysis be made in Drosophila melanogaster?

I've asked this before in stack overflow's cognitive science community, and someone recommended me to ask here: I've found a couple of studies using microdialysis on insects, but didn't found any in ...
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3answers
5k views

What is the advantage of indirect ELISA over direct one?

I guess the answer is about indirect one giving less error due to selectivity but how exactly does that happen?
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1answer
940 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 ...
2
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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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1answer
484 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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0answers
70 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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2answers
440 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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2k views

How does air-liquid interface (ALI) culture work?

For studying diseases, such as chronic obstructive pulmonary disease (COPD), cells are often grown in air liquid interface. I understand that the common way of establishing this these days is to grow ...
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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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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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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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1answer
3k 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
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
734 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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3answers
200 views

Extending a small fragment of DNA

Is there a way to extend a small fragment of DNA, say 150 bp, by making copies of itself and attaching each copy of that small fragment to the end of that 150 bp sequence? For example, I want a 1 ...
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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
274 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
108 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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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
438 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
757 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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0answers
113 views

Effective Mycoplasma Elimination from Primary Human Cultures

Obviously the best way to avoid mycoplasma contamination is to avoid it in the first place. In our case, however, it is not possible to avoid. We are culturing viruses and tissues out of human nasal ...
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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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4answers
866 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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2answers
1k views

What is the importance of urea in mass spectrometry?

What is the importance of urea in mass spectrometry? We use 8M urea to FASP our proteins prior to mass spectrometry. What is the significance of using 8M urea? and how does it affect the proteins?
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31 views

Extraction of arthropod embedded in animal tissue

I'd like to extract an ectoparasite from a biopsy of animal skin for subsequent identification. The mite is small, about 100 um, and embedded in a column of tissue. It's too small to manually excise ...
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1answer
2k views

Extracting cell-free DNA

Has anybody had any success in extracting cell free DNA from plasma without using expensive kits? I've already spent a lot of my own blood trying to use standard phenol/chloroform methods with very ...
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1answer
392 views

Do bacteria grow on pure dry glucose?

I've accidentally touched pure glucose with my bare hands (fingers to be specific), which was intended for cell-culture. I'm worried that bacteria from my skin may start to grow on the glucose and ...
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1answer
393 views

Respiration of an animal cell media

I want to do respiration process of an animal cell media in laboratory environment. I don't entirely sure how to supply it with Oxygen to the process because it is a gas, but not a liquid.
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1answer
377 views

Random Mutagenesis vs Directed Evolution as Strategies to boost expression

Do people use random mutagenesis (say using UV) to generate host variants that have high expression of a metabolite / enzyme? I've seen it mentioned as a strategy but it confuses me as to why. How ...
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1answer
40 views

Voltage sensitive dyes technique: What is the underlying measure?

I just discovered voltage sensitive dyes technique: first of all what imaging techniques do we use? And I have seen that figures are labeled with ΔF/F0, what does it stands for?
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1answer
255 views

Does adding antibiotic after 5-10 mins of innoculation affect the protein yield or growth?

I've asked a lab colleague the same question. She said, it would loosen the bacterial cells in the LB medium and plasmids would come out. Is that true? and why?
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1answer
2k views

Optimizing Gel Electrophoresis: Ampere, Volts and Buffer concentrations

I am a master student in biochemistry, and I have used gel electrophoresis many times before. What I want to know is how one should adjust the mA (mAmpere) compared to the voltage and the buffer one ...
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2answers
411 views

Measuring fitness / lifetime reproductive success (LRS) in Drosophila

I am planning a fitness assay of Drosophila melanogaster. I'd like to get a good measure of lifetime reproductive success (lrs) but I don't want to count all the offspring produced over a lifetime by ...

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