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

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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
30 views

How to double check plasmid detected by PacBio?

We detected a 50 kb sequence of plasmid apart from the chromosomal sequence by whole genome sequencing. We are interested in wet lab validation of this plasmid for publishing. Even though wet lab ...
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3answers
153 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
58 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
15 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
32 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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8 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
15 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
31 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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47k views

Why do we add salt when precipitating DNA?

All the DNA extraction protocols I have seen involve adding salts to the extraction buffer. What is the purpose of the salts? What happens if they aren't included?
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93 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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1answer
31 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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8 views

What steps need to be taken between a successfull SDSPage GE and MS/MS?

Once you have your gel with separated proteins can you simple cut out each band and plop it into solution or do you need to take extra steps?
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1answer
26 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 ...
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1answer
27 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 ...
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3answers
51 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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2answers
33 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
18 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
37 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 ...
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1answer
188 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
432 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
352 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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20 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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555 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
64 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
26 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
27 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
23 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
55 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
55 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
234 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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1answer
34 views

Pooling for qPCR

I am comparing miRNA expression levels in 3 different groups but I am low on money and time. I have to get some preliminary results to get the actual research going so I decided to pool my samples and ...
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0answers
11 views

Stable isotope sample preparation: Bone Collagen

I am performing research in which I need to extract bone collagen from the foot bones of American Beaver (Castor canadensis). I have read through the literature fairly thoroughly on both the ...
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1answer
132 views

Help analyzing SDS-Page gel

In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal ...
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27 views

Alternative to Sterilizing Under Pressure

I want to grow mycelium in agar, and all the resources I have found say to sterilize the agar under 15 psi, but I do not have the necessary equipment to sterilize the agar under pressure. Is there an ...
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2answers
92 views

Getting PCR amplification at annealing higher than Tm!

I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong ...
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41 views

My flow cytometry antibody won't reach saturation

I am staining human neutrophil populations (CD177) and then trying to get a different test target antibody to give a dose response to binding to this population. I have tried concentrations of the ...
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1answer
150 views

Good pipetting technique?

Good pipetting technique is essential for many biologists, but it can be hard to get right. When I take 1 µl of liquid using a micropipette, I seem to always take less than 1 µl, and that amount is ...
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0answers
18 views

How would one identify cellular transcription factors associated with a viral protein in a treated cell line?

I've been working as the computer guy for a microbiology lab for the past few months. I've always been interested in bench work, but my wet lab experience is rather limited and thus so is my ...
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1answer
45 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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3answers
538 views

Amplification technique for proteins similar to PCR for DNA?

I know PCR can be used to amplify a tiny sample of DNA in order to perform experiments. Is there a similar technique to use on a protein sample? More specifically, I'm not interested in "cutting" up ...
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50 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 ...
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63 views

Making mistakes in the laboratory [closed]

I have just started an internship in a microbiology laboratory. I have had a rather small amount of laboratory experience before this so had to learn mostly everything from scratch. I have been there ...
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1answer
592 views

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 ...
3
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1answer
50 views

Resolution of X-ray crystallography

A structure determined by X-ray crystallography has a resolution of 1.5 Å. When I look at the coordinates, I find every backbone C-N distance is 1.32 Å.i.e. Accurately predicted. If resolution is not ...
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1answer
5k views

When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
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22 views

What Helminth species has the simplest LABORATORY CONDITION for hatching?

I am very interested to hatch the eggs of a parasite in LABORATORY ! but for my first experience I need a species that is not so expensive or time consuming for experiment and can be done by an ...
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0answers
54 views

Why do I get such a strong background in a detection of DIG-labeled DNA (Southern Blot)?

I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin ...
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139 views

In CRISPR bacteria, how does viral genomes get integrated into the spacers of CRISPR? Also, in its use, where does Cas9 cut the DNA?

I've been out of Biology for about a year polishing my programming skills. I know CRISPR/Cas9 allows targeted 'cutting' of DNA via RNA-guidance. Few questions regarding this. Regarding to its ...
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Why do PCR manufacturers recommend assembling the reaction mix on ice?

Many PCR manufacturers recommend preparing the reaction mix for the reaction on ice. For example, here is NEB's recommendation for their Q5 polymerase. Other manufacturers also include similar steps: ...