Questions tagged [lab-techniques]

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

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0answers
24 views

who knows what is this picture? [closed]

Can anyone try to help me identify this picture? Thanks!
2
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0answers
35 views

Does glycerol in E.coli culture media somehow inhibit the lac-operon?

I have have been taught that one should induce protein expression with IPTG at an OD of about 1.0 - 2.0 when E.coli grows it TB media (terrific broth). As a reference point, one typically induces ...
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1answer
39 views

bunsen vs meker for best sterile field [closed]

I need one bunsen and i have a friend that sells me a bunsen-meker burner. I see that bunsen-meker burners have more flame diameter, so should have more diameter of sterile field, but i dont know XD ...
2
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1answer
100 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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0answers
33 views

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 ...
5
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1answer
27 views

Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
1
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1answer
17 views

Low-tech or low-cost technique for quantitative estimation in enzymology

If an accurate measurement of enzymologic quantities is needed, then following established methods in the field is necessary. However, it is sometime of great usefulness to ballpark a value before ...
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1answer
36 views

Book recommendation: protocols and recipes handbook for molecular biology / biochemistry

I'm a 5th-year PhD student in chemical biology. I've mostly been doing computational work, so my bench skills are rusty. To help me, I'd like a handbook of common techniques -- transformations, ELISA, ...
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1answer
43 views

Coronavirus and temperatures

An article on bioRxiv, Evaluation of heating and chemical protocols for inactivating SARS-CoV-2, recommends certain treatments to inactivate SARS-Cov-2 for lab work. The abstract notes: "Although ...
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3answers
2k views

Why does an autoclave need to pressurize steam?

I've looked this up on other sites and they say that an autoclave needs high pressure in order to raise the boiling point of water so that you can achieve steam of a higher temperature. If you look ...
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0answers
24 views

Can I use a Flask Weight Ring to increase RPM in an orbital shaking incubator?

I am working on a device that goes on top of the flask. This reduces the maximum RPM that I can use. Therefore I am wondering if I can use a Flask Weight Ring for Erlemeyer flasks in a orbital ...
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1answer
19 views

How is in-vitro fertilization technically achieved?

I failed searching on Google technical details about how in-vitro fertilization is achieved. I'm referring to the toolset and its use, rather than the chemical or biological aspects of the process. I ...
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1answer
30 views

How does the choice of blood draw site influence the possible specificity of a serological test?

The news has reported that a new serological test for the presence of anti-SARS-CoV-2 antibodies has received an emergency use authorization from the FDA, and notably has a higher specificity than ...
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33 views

qPCR to detect viral load

In an experiment qPCR was used to determine viral load. Alongside this conventional PCR was also performed and ran on an agarose gel. However I do not understand why both methods would have been used, ...
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0answers
17 views

How do you sanitize blood for use in a BSL-1 lab?

I do research in a BSL-1 (biosafety level 1) lab, though I have access to a BSL-2 lab if needed (though very inconveniently). I'm doing medical research with the goal of identifying biomarkers for ...
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19 views

Using a magnetic stirrer as a makeshift shaking incubator?

I'm working in my high school's lab and need to culture bacteria in a broth culture. More specifically, I'm trying to harvest Bacillus endospores with the exhaustion method published in "Molecular ...
2
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1answer
116 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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0answers
12 views

How to choose an assay for detecting virulence factors?

When looking for a protein of interest, an ELISA is being administered. I read this article about the different types of ELISAs and the advantages/disadvantages. However, I am conflicted about ...
3
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2answers
62 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 ...
2
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0answers
38 views

RNA product storage in isopropyl alcohol

I have an issue. I isolated RNA from cortex a few days ago. For isolation of RNA i used this procedure: I homogenizised my sample (mouse cortex) with Trizol. Then to the sample add chloroform, shooke ...
0
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1answer
19 views

Minimum amount of RNA for cDNA conversion and qPCR

I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased ...
0
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0answers
28 views

What is the most durable electronic pipette?

We have a routine task of pipetting substance concentration series in triplicate on cell cultures under a laminar hood. To speed up the process, we use an electronic pipette in the repetitive ...
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0answers
18 views

Are controls needed for urine cultures?

If testing for bacteria (gram-negative) in a urine culture, are negative or positive cultures needed? I could not find anything that says controls are needed unless results seem to be contaminated. ...
0
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1answer
98 views

Lab technique to distinguish between single stranded and double stranded DNA?

What lab techniques exist to differentiate between single-strand and double-stranded DNA?
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1answer
94 views

Why spin live fruit flies in centrifuge?

I was reading this article about the evolution of monarch butterflies’ resistance to cardiac glycosides: These Butterflies Evolved to Eat Poison. How Could That Have Happened? And this passage made me ...
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2answers
54 views

Bacterial Growth Formula

What are the units of $N$: the colonies of bacteria or the viable cell count? This is in regards to the bacterial growth formula used during serial dilutions, $$ N =N(0) e^{kt} \quad . $$
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1answer
64 views

Autoclaving media Question

1.When I autoclave media should I close the cap tightly or slightly? I put all tools in plastic bag when I autoclave, should I close slightly the plastic and when it finish I close tightly the ...
4
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1answer
200 views

Why is sorbitol used in buffers?

Many protocols in my lab use sorbitol in buffers. For instance, in co-immunoprecipitation, we include it at a final concentration of 200 mM in our lysis buffer. I'm not entirely sure why though. I ...
1
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1answer
75 views

How can I improve efficiency of Ecoli transformation?

I am an intern in biology institute. I have a 17.3kbp plasmid need to transform to Ecoli. But I have tried many time but have no or very few colony on LB plate. I know large plasmid have less ...
3
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0answers
29 views

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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2answers
320 views

Why do protein solutions have to be alkalised in biuret test?

I’ve read that CuSO4 solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I ...
3
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1answer
87 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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2answers
59 views

Preparing sample for SDS PAGE

I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way ...
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2answers
64 views

I did an agar gel electrophoresis but I could not see DNA, why? [closed]

After performing a PCR, I ran the products (700 bp) in an agar gel electrophoresis along with a Genedirex ladder (100 bp), but the transilluminator did not reveal them.
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19 views

How do I homogenise a small pellet of 10 to 50 cells within less than 50 µL?

I have a problem with declumping/ homogenising bacterial cells in small volumes (10 to 50 µL). I know that I can get all of my cells out of the tube, but they are still stuck together and won't form ...
0
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0answers
15 views

Re-stain slice using a different secondary antibody?

I stained a free-floating ~300uM brian slice using 4 different 1º antibodies (rabbit, chick, mouse, rat). For the mouse primary, I mistakenly used an Alexa-350 secondary, when I should have used 594. ...
2
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0answers
13 views

How to isolate a high proportion of available helminth ova from animal faeces/feces without damaging them?

I need to isolate large numbers (tens to hundreds of thousands) of cat (and later dog) hookworm ova from cat/dog faeces. I have a centrifuge, and various sieves with pore sizes of 20, 63, 75, 106, ...
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 ...
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0answers
27 views

Why both forward and reverse primer are added in asymmetric PCR?

If the purpose of Asymmetric PCR is to amplify single targeted strand of dsDNA then instead of adding just one primer for targeted strand why do we add both forward and reverse primer? Upon addition ...
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0answers
14 views

Does the preservation fluid (ethanol or formaldehyde) leach heavy metals from mollusc tissue?

In a thread on researchgate, the concern was raised that long term exposure to unbuffered preservation fluids such as ethanol or formaldehyde may leach heavy metals from biological samples due to a ...
6
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0answers
70 views

What is the fastest way to crystallise lysozyme (for student course)?

High school sudents are going to visit my university and I plan to demonstrate crystallisation of lysozyme. I ordered pure lysozyme from VWR. I can easily crystallise this within 15 min in batch (4% w/...
5
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3answers
205 views

Double Digestion with Restriction Enzymes Using Different Buffers

I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site ...
2
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1answer
130 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 ...
0
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0answers
34 views

Is it normal to see yeast cells stacked in the Z axis when counting yeast?

I just got started with yeast cell counting and was surprised to observe yeast cells that are at different z axis depths at 400x. That is, by adjusting the focus (not the x/y stage) I can make some ...
0
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1answer
32 views

How to confirm secondary structure formation of Precursor miRNA on gel?

I want to fold precursor-miRNA into its secondary structure and then confirm it on gel. At first, I heat it in annealing buffer at 95°C for 5 minutes and cool down slowly. Then I run it on gel but the ...
1
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1answer
160 views

Killing microorganisms on agar plates?

If I grow microorganisms on agar plates and I expose them to UV light. How exactly will I know that the microorganisms have been killed? I was watching a video that showed spots in the petri dish ...
0
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1answer
52 views

How many cells should be seeded for passaging?

Do you count cells when you passage them? I found that many people will according to a ratio (maybe 1:2 or 1:3,etc.) to subpackage. It is so indistinct! What is the optimal seedling density or ...
0
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1answer
48 views

Improving DNA quality and yield from stool samples

I prepared genomic DNA from pig stools but the concentration was very low — about 3~4 ng/µl, when I measured it using nanodrop. The A260/A280 and ...
1
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1answer
66 views

Why pellet and resuspend E. coli for plasmid prep

For maxipreps, cant you just add all the stuff that would be in P1 (RNase A, EDTA) then just add P2? Because if we pellet cells then resuspend theres got to be a reason, right? Is it slats and other ...
2
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1answer
57 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 ...

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