Questions tagged [lab-techniques]

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

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53 views

DNA preservation at room temperature

I am considering preserving the DNA of a family member who passed away. The funeral home offers a service called DNA Memories from a Canadian company (CG Labs). They have two options: Store the DNA in ...
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47 views

Can anyone expolain this? Cell culture vials have strange hair-like condensation patterns

What is the hair-like polymer that condenses on the caps of the plastic cell vial? Inside vial are cells in a mixture of FBS and DMSO. The vials were frozen slowly in a special isopropanol-filled ...
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35 views

How do I test if microbes have survived after dehydration?

I have a solution containing various bacteria and fungi. My aim is to place solution on filter paper, and wait until it dries. I then wish to test if the organisms have survived, either on the dried ...
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0answers
622 views

Origin of the 260/280 ratio?

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, ...
3
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264 views

Gibson assembly using NeBuilder

I am supposed to construct a plasmid that contains features from two other plasmids. My strategy is to generate three fragments form the two other plasmids. I was encouraged to try Gibson assembly, ...
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0answers
482 views

Resuspending Cells from a filter plate

My experiment involves pulling down cells on to a filter plate for a variety of assays. However, I want to ensure that my cells are alive so I am trying to resuspend them so that I can do an accurate ...
2
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1answer
3k 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?
2
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1answer
150 views

1% water solution of deoxycholic acid. How is it prepared? [closed]

According to Sigma, the solubility of deoxycholic acid is: 0.24 g/L in water at 15C According to the FDA, Kybella (trademark) is a 1% water solution of deoxycholic acid. How did they do this?
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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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3answers
2k views

Is RNase AWAY in the lab dangerous?

I use RNase AWAY in the lab. I would like to know how dangerous this chemical is for health. For example, when I remove my gloves my hands smell because of the RNAse AWAY
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2answers
696 views

How to separate peptide on SDS PAGE?

I want to separate 1600 Da peptide on SDS PAGE, on 20% GEL I didn't get any band, How can I separate on PAGE? Can I use 25% SDS PAGE?
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1answer
78 views

Decreasing signals in assay measurements

I'm working with a calcium assay to study the effects of different virulence factors. The assay works, but from day to day the signals of cell lysis go down. Unfortunately, I haven't found an ...
2
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1answer
130 views

How does SDS-PAGE separate based on mass?

In SDS-PAGE, electric field and mass-to-charge ratio are approximated to be constant for all proteins. Also, if $F=qE =ma$, then $\frac{m}{q}a=E$. Thus, all proteins must migrate with a constant ...
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2answers
557 views

Why do many DNA solutions contain additional compounds?

DNA solubility data in only water is scarce. A previous question asked for a quantification of DNA solubility in water. It seemed like it would be easily answerable, however isn't quite that simple ...
2
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1answer
378 views

Substitute 25mM dNTPs mix with 10mM dNTPs

I need to make a solution of multiple compounds, one of them is dNTPs. The recipe calls for 20 μl 25 mM dNTPs in a 1250 μL master mix. Unfortunately I do not have it available at that concentration, ...
2
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1answer
81 views

How can enzymes be immobilised on glass?

I’m studying a hypothetical model for urease activity, which involves fluorescence measurement, hence the need for an optical window to which the enzyme urease is immobilised. From my previous ...
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1answer
1k views

Why shouldn't I clean microscope slides with a paper towel?

I've recently bought a microscope and been looking at a lot of bacteria and such, and have quickly run out of clean slides. I've seen videos and articles saying how you shouldn't clean microscope ...
2
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1answer
32 views

What do the column labels in this table mean?

This table shows the effect of sound (song and tones) on female birds. However, I'm not sure what the labels (F1 22, P and η2) mean. I've seen the labels on other tables too. (from https://www.ncbi....
2
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1answer
742 views

Copper for cell incubator to prevent contamination

For some reason the lab seems to have a problem with contamination every so often. It's virtually impossible to prevent bacteria, viruses, fungi, etc. from getting into the incubator every time you ...
2
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1answer
32 views

What does it mean to “cure” microscope slides?

I am reading a journal paper and I have come across the following statement in the materials and methods section regarding the preparation of microscope slides: Coverslips were washed once more with ...
2
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1answer
65 views

Why electroporator need a cuvette?

I have never obtain an Electroporator for my home-lab. Because it's very expensive, I'm trying to create a diy cheap version for myself. In all commercial design, I see that we need to put the sample ...
2
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1answer
97 views

When in Ampicillin degraded (gone) in liquid TB-media? Concerns about selectivity

Question: Specifically regarding Ampicillin; When growing cells in TB (terrific broth) for protein expression - when should I expect the ampicillin to be gone due to degradation by b-lactamases? (and ...
2
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1answer
320 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
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1answer
839 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
111 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 ...
2
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3answers
4k 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 ...
2
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1answer
179 views

Do I have to use sucrose to induce a lac promoter?

I'd like to optimize the expression of a Fab fragment in Escherichia coli. For induction of the lac promoter on the pAK400 vector I use IPTG and sucrose. Do I optimize the expression in case I would ...
2
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1answer
91 views

Do you have experience with PacBio?

I prepare a experiment and I found $PacBio SMRT$ as the great way to sequence my PCR products. I find the cost: library preparation 655 dollars + sequencing 435 dollars. It seems very low. Do you have ...
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1answer
128 views

Pippin prep kits - expiration date

Do you use Pippin prep? We would like to buy it, but we need more info. What is the expiration date of kits for Pippin prep? Thanks!
2
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1answer
2k 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 ...
2
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1answer
46 views

Why when measuring turbidity do we use the minimum wavelength?

As a preface, I read a few other related posts and was able to gather some knowledge, though without any background in physics I am having some trouble here piecing together a coherent view. I looked ...
2
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1answer
18 views

How to supply oxygen to a culture vessel?

I'm reading this paper: https://www.jstage.jst.go.jp/article/jpsa/51/3/51_0130043/_pdf From day 17 of the culture period until hatching, pure oxygen was supplied at a flow rate of approximately 500 ...
2
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1answer
144 views

Must I HPLC-purify my PCR Primers for amplicon sequencing with PacBio SMRT?

I would like to order some primers for amplicon sequencing. I am using universal tag primers (unusually long 30-mers recommended by PacBio for SMRT, both F&R) + barcodes (16-mers). The resulting ...
2
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1answer
184 views

Help with preparing Congo Red TSA

So I want to make Congo Red TSA. My instructor gave some hints but I still have some questions: I start by making two stock solutions: 20 mg/ml of congo red and coomassie in two different falcon ...
2
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1answer
154 views

Is it possible to produce a tangible mass of DNA/RNA using a PCR reaction?

A Polymerase Chain Reaction (PCR) is a method of amplification which enables one to produce many copies of a certain DNA/RNA strand for many applications (as described in the link above). I have ...
2
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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 ...
2
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1answer
21 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.
2
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1answer
44 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?
2
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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 ...
2
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3answers
615 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 ...
2
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2answers
225 views

Career progression through biosafety levels?

Does a career in infectious disease typically progress through biosafety levels, or do people select one and specialize their training in procedures specific to those hazards? That is, say Dr. ...
2
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1answer
137 views

Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?

I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-...
2
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1answer
2k views

Does methanol fixation deform cultured cells?

I use methanol fixation (@ -20⁰C for 10 minutes) when performing immunofluorescence assays on cultured cells. Generally speaking, this results in very good antibody staining. However, the cells tend ...
2
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1answer
64 views

How can I view modENCODE data faster?

I am trying to view several data tracks in the modENCODE GBrowse genomic browser. However, the site is so slow, it is practically unworkable. Is there a faster way to explore the data?
2
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1answer
520 views

What's the difference between a free chromosome fragment and an extrachromosomal array?

This is reference to a review on C. elegans mosaic analysis by Yochem and Herman, in which the authors make a distinction between free chromosome fragments and extrachromosomal arrays. For the former,...
2
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1answer
155 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 ...
2
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1answer
964 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 ...
2
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2answers
89 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 ...
2
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1answer
928 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 ...
2
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1answer
334 views

Where does the inverse seconds unit come from in the association constant?

I'm working to determine Kd(s) kinetically by generating association and disassociation curves. Kon (association constant, or on-rate) is in inverse seconds multiplied by inverse molar. I get that ...

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