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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9 votes
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What is the purpose of using two layers of gel in SDS- PAGE?

The stacking gel concentrates proteins loaded into the sample wells so that they are resolved as a unified "line" once they enter the stacking gel. The reason for the lower pH is that this "lower ...
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  • 636
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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7 votes

Machine learning for light microscopy -- problems to solve?

I know this question is going to close. But, if you want to work something you can work on: Cryo super-resolution fluorescence imaging Highlights CryoFM allows imaging of vitrified ...
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7 votes

Pipetting: do human experimenters need liquid class information?

Just to be clear, it has been many years since I have done real lab work, so I am certainly not an expert on the specific question. However, I have a fair amount of experience writing clear ...
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  • 71
6 votes
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Can bacterial RNA be degraded at -80⁰C in a SDS solution?

I think it is relatively unlikely that the RNA will degrade under these conditions. For the future, I would handle this differently: You can centrifuge the cells and snap freeze them in an ...
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6 votes

What is the purpose of using two layers of gel in SDS- PAGE?

The other major difference between the two is the amount of acrylamide in the upper (stacking) gel - it's generally around 4%, while the lower (resolving) gel can vary from 6 or 8% to 20%, depending ...
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6 votes
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How can I convert plate reader measurements of Pichia pastoris OD to cells per ml?

From a technical perspective, for the FlopR package you mention (and I believe also for Jacob Beal's protocol) you can simply change the bead size and perform a new calibration. The only information ...
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5 votes
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Time required for RNA precipitation in ethanol

As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). ...
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5 votes
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Function of heparin and dextran sulfate for removing proteins

See here. Histones are basic proteins (cationic, high pI) because they are required to interact with polyanionic DNA at physiological pH. Heparin and dextran are polyanions which form insoluble salts ...
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5 votes
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Changing time and rpm of centifuge

No, it isn't that simple, because centrifugal force varies with the square of the rotor speed. Have a look at the Wikipedia page for clearing factor. Those equations are usually used to determine ...
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5 votes
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Explanation of protocol in DNA extraction experiment

The DNA solution has a fixed volume (e.g., 0.1 mL). To a suitable test tube is added 0.2 mL of buffer-saturated phenol. After closing the test tube the mixture is mixed well, typically using a vortex ...
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5 votes
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How do you keep track of past/new lab protocols?

The best match to what you're looking for is probably https://www.protocols.io/, which hosts protocols for free and generates a digital object identifier (DOI). It is not indexed by PubMed or other ...
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  • 3,338
5 votes

Is it possible to use PCR to test for Machado-Joseph disease?

This paper describes an allele-specific PCR protocol to detect CAG repeat expansion in MJD patients: Maciel P, Costa MDC, Ferro A, et al. Improvement in the Molecular Diagnosis of Machado-Joseph ...
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  • 7,214
4 votes

Machine learning for light microscopy -- problems to solve?

RE: What kind of software you always wanted for light microscopic research, but did not know how to build? I research fruit flies and in this field (and many other insect ecology model systems like ...
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4 votes
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Why extract DNA from certain white blood cells instead of whole blood?

There are several reasons why a lab might choose to get DNA from lymphocytes instead of whole blood. Generating genomic DNA from whole blood is not necessarily the best idea, as all the excess protein ...
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4 votes

Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

There are already many great answers to your question, however I thought I put my comments in form of an answer. The standard for DNA agarose gel is TAE and for the protein, it depends on the size of ...
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4 votes

Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

Grossly, it does not matter what buffer you use. It is the pH that matters. For DNA electrophoresis EDTA is added in order to chelate divalent cations that serve as cofactors for nucleases. Tris is ...
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4 votes
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Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them: TBE is a better conductor and is thus less prone for overheating the gel Borate is a ...
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4 votes

Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

I have had good experience using a lithium boric acid buffer from Faster Better Media. I use it for RNA gels, but it's advertised for DNA gels. I don't think it can do protein, but I've never tried it....
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  • 5,288
4 votes

Is a sequential double transformation acceptable?

If the plasmids you wish to transform are very big (more than 20kb), the co-transformation might not be very efficient (you might not get any colonies). Electroporation should work though, as ...
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4 votes

Why is it sometimes difficult to resuspend E. coli in P1?

Some things to consider. If you spinning too fast and too long, that is going to pack the pellet more. You can spin longer at a slower speed and you will notice your pellet is not as tightly packed. I ...
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4 votes

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

What I find works is to scotch tape the labels to the tubes, they hold up fine in our -70C freezer. What I did was go into excel 2010, go to View tab, click on Page Layout on the left side, not ...
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  • 5,288
4 votes
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How does one experimentally obtain a hemoglobin-oxygen dissociation curve?

There are existing instruments commercially available to measure hemoglobin-oxygen saturation curves. Whole blood or lysed blood can be used; I would expect the instrumentation to come with specific ...
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4 votes

Storage DNA Condition

Should be fine, but if I were you, I'd spare a few more minutes and carry on until ethanol precipitation (15 min of work at worst). Here's the protocol: https://www.thermofisher.com/pl/en/home/...
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3 votes
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Can I use grayscale images when working with ImageJ?

There is no evidence that one is better than the other, most likely because it differs from case to case. Neither you, nor your critics, are right. There is a tiny bit of science in a paper on ...
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  • 1,120
3 votes

What's up with all the vague protocols?

This is too long for a comment, so I post my thoughts about this here: This falls into different important parts here: One is an insufficient understanding of the techniques on which the method is ...
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3 votes

Change solvent of bacterial solution

I'm assuming that you have a solution of bacteria in your buffer directly after growing them for some time, and without performing a lysis step yet. The 2% SDS step looks like it is for lysis of your ...
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3 votes
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What is "bacto" peptone?

See here. Bacto is a brand which used to be marketed by Difco. It is a casein (milk protein) hydrolysate.
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