29 votes

Why does an autoclave need to pressurize steam?

An autoclave can sterilize both solids and liquids, whereas an oven with no pressure control is typically not suitable to sterilize liquids. Not only do you need to heat the chamber to 121°C, but you ...
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  • 1,883
26 votes

How long will a typical bacterial strain keep in a -80°C freezer?

Richard Lenski's lab at Michigan State university has been doing long-term evolutionary research on E. coli and maintaining a "frozen fossil record" of their experimental populations every ...
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  • 4,496
23 votes
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Pipetting: do human experimenters need liquid class information?

In my experience it is very rare to see a protocol for humans that describes how to pipette a certain liquid, but I don't have as many years pipetting as others do. In general, it is left to the ...
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23 votes

Pipetting: do human experimenters need liquid class information?

I work on software that controls one model of robotic pipetter. There are a few reasons why we use complex liquid class definitions: One of the selling points of using a robot is high speed. You ...
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15 votes

Are bleach solutions still routinely used in biochemistry laboratories to rid surfaces of bacteria, viruses, certain enzymes, and nucleic acids?

I'm a research post-doc working in a US-based microbiology research lab. We still use a relatively low-concentration bleach solution to decontaminate most liquid cultures before disposing of them, but ...
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  • 4,496
13 votes
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How sterile is sterile when working with nucleic acids to prevent contamination?

Short answer: Yes Long answer: Depends what you are working with. DNA: If you are working with DNA, its pretty stable and you can usually get away with a 70% ethanol wash/autoclave (mainly to ...
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  • 2,268
13 votes

When did mouth pipetting stop becoming a way to handle liquids in a lab?

Mouth pipetting, while almost unheard of in modern laboratories in developed countries, is still very much a current protocol in many parts of the world. For example, this paper analyses the ...
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  • 9,304
13 votes
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Assembling small DNA parts using Golden Gate

Purely going off experience here having used golden gate assembly methods for 5+ years now, there is a definite lack of literature regarding small part assemblies. In my current lab, we use CIDAR ...
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  • 316
12 votes

Are there issues with filling PCR tubes to capacity?

Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to ...
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  • 49.1k
12 votes

Are there issues with filling PCR tubes to capacity?

(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki ...
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12 votes
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Why are cell lines frozen in vapor phase?

The storage itself can be done both in the liquid and also in the vapor phase in the liquid nitrogen. Both have their advantages and disadvantages. In my experience there are two main reasons why ...
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12 votes
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What is the role of glucose in plasmid isolation?

This is a common misconception: in fact, in the alkaline lysis method for plasmid isolation, the glucose does not act as an osmotic stabiliser. Glucose is present in the resuspension buffer at 50 mM. ...
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11 votes

Good pipetting technique?

Here's some step-by-step advice on good pipetting technique: Make sure the pipette is set for the correct volume Ensure the tip is firmly attached Keep the pipette vertical when pipetting Slowly and ...
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  • 16k
11 votes
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How long will a typical bacterial strain keep in a -80°C freezer?

The reason you haven't found a definitive answer is that it depends on several factors. The first factor that comes into play if we consider only ultra-low freezers (- 80 °C) is the organism you're ...
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  • 466
11 votes
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What's happening in the "C" and "T" stripes of a covid test kit?

This is a lateral flow assay. [image source] A sample is applied at one end of the strip and flows across to the other side by capillary action. It first encounters antibodies against the target ...
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10 votes
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How to avoid air bubbles while pipetting?

You're still pipetting too fast, you should move the piston slowly and evenly to avoid air bubbles. Also make sure that you wait a second or two for the liquid to rise before moving the tip out of the ...
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10 votes

Should I dilute DNA with water or elution buffer?

I would generally recommend using the elution buffer (which is typically Tris-EDTA buffer) or TE-Buffer, as the pH and the conditions stabilize the DNA for a longer time, which is especially ...
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  • 49.1k
10 votes

Are bleach solutions still routinely used in biochemistry laboratories to rid surfaces of bacteria, viruses, certain enzymes, and nucleic acids?

I can only speak from my experience in many labs in different countries: We never used bleach for cleaning or disinfecting surfaces like cell culture hoods or lab benches. For this purpose we ...
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  • 49.1k
10 votes
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Saturated Mutagenesis Screening

This sort of method is indeed quite useful and frequently used in synthetic biology: I've used a similar approach before to generate 5' insulators for promoters. Calculating the exact theoretical ...
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10 votes
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Tips for longer fragment size and higher purity of insect DNA

Insect samples are difficult to work with primarily because they contain a bunch of polysaccharides, like chitins, that are similar enough to nucleic acids that they can co-precipitate. Standard ...
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  • 6,278
9 votes
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Gloves for Cell Culture?

In my opinion wearing gloves during cell culture is a good idea. It works both ways as a protection: For your cells since you are not loosing small danders from your hand and for you since you are not ...
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9 votes
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How do I put on a labcoat?

Wear two sets of gloves. First, use nitrile gloves, not latex for this. 4mm or thicker is a specification I would recommend to avoid tearing. Latex will bind too well to itself and tear easily with ...
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  • 4,089
9 votes

How sterile is sterile when working with nucleic acids to prevent contamination?

First of all, sterility is not necessary. It takes much more effort to reach this than just to wipe down everything with ethanol. What you need is a clean and controlled work environment (but this is ...
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9 votes
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"X" in stock solutions

1x is the final working concentration of the solution (it could be anything depending on the type of the solution). Stock solutions are made at a higher concentration; if it is 10 times more ...
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  • 35k
9 votes
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Why spin live fruit flies in centrifuge?

Correction of Poster’s Source The article cited mistakenly refers to centrifugation, however the actual procedure used in the work — Karageorgi et al. Nature, 2 Oct 2019 — is vortexing, as ...
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  • 22.3k
9 votes

Alternative dyes for Gram staining

As you may have realized, crystal violet can be replaced by a lot of dyes since the ethanol will wash out the dye from the gram negative cells. Methylene blue is a nice choice - malachite green may ...
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  • 201
8 votes

How can I label cryotubes in a way that eliminates the problem of legible hand-writing?

This is indeed a problematic point. I have been using machine printed labels for a while now (sometimes with some extra scotch tape around it to prevent it from falling) off, which worked pretty ...
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  • 49.1k
8 votes
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Magnetic-activated cell sorting vs. FACS

Magnetic-activated cell sorting (PDF download), or MACS, is a procedure developed by Miltenyi Biotec to separate cells from complex mixtures using antibody-coated magnetic nanoparticles. The ...
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  • 14.6k
8 votes

Measuring luminescence in a fluorescence plate reader

I am René Inckemann and I am PhD student at the Max Planck Institute in Marburg, Germany. In my PhD I am using luminescence on a regular basis and I was also responsible to organize some of our ...
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8 votes
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Measuring luminescence in a fluorescence plate reader

As the other answers here say, technically plate readers which are capable of fluorescent measurements can also make luminescent readings, but the sensitivity may be low (https://www.biotek.com/...
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