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 ...
Dan Bryant's user avatar
23 votes
Accepted

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 ...
Noah Sprent's user avatar
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 ...
apc's user avatar
  • 71
6 votes
Accepted

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 ...
ajhfedorec's user avatar
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 ...
acvill's user avatar
  • 8,296
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 ...
arboviral's user avatar
  • 3,344
4 votes
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Effect of ethanol on TRizol RNA extraction

My answer is based on my experience in the lab and something like a best guess - it may or may not work. But given the fact that added 100µl of ethanol to 500µl of total volume and since water and ...
Chris's user avatar
  • 51.6k
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 ...
Bryan Krause's user avatar
  • 45.7k
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/...
Krzysztof Czarnecki's user avatar
3 votes

Improving contrast between dot and paper in dot blot

In the absence of another answer, I'll transition my comments to an answer. Please label your images. Both an Abcam guide and a Thermo troubleshooting document suggest using a kit or DAB protocol ...
Maximilian Press's user avatar
2 votes
Accepted

In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up?

The HRP is an enzyme so it isn't a component of the reaction. By definition, enzymes only catalyze reactions but aren't an actual component of the reaction. As such they don't get used up during the ...
yp66t89's user avatar
  • 226
2 votes
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Why is it good practice to prepare western blot samples in pairs when you have two experimental groups?

This point is not unique to Western Blot, but to any case-control study: if there is variability inherent in the preparation procedure, this variability will be reduced when the two samples are ...
Roger V.'s user avatar
  • 3,852
2 votes
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When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?

It is because you don't know how your treatments affect the expression of the total specific protein; perhaps it down- or up-regulates the amount of protein being made, but the relative proportion of ...
bob1's user avatar
  • 12.1k
2 votes
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Should I use RNAseZAP on my bench and pipettes before RNA extraction?

The common practice in my lab is to work in a laminar flow cabinet and clean it with alcohol, then spray it with the RNAseZAP. I've always heard from my professors that, while DNAses are more uncommon,...
LinuxBlanket's user avatar
  • 1,313
2 votes
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Why marker and control are loaded in the beginning or ending lane of the gel?

No important reason. It is for esthetics, and also for ease of description in figure legends. One could argue that it might be confusing to have the markers or the control in the middle, but since the ...
Karl Kjer's user avatar
  • 7,663
2 votes
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Reversal of cross-linking in ChIP-seq

After the crosslink reversal step, you still have some DNA connected to protein fragments. So at that step, instead of doing a normal DNA purification, we also need to use a phenol-chloroform ...
julianstanley's user avatar
2 votes

Pipetting: do human experimenters need liquid class information?

In my experience, I've never seen more than "pipette 100 μl" or at most "caution viscous liquid". Also I've only been taught how to pipette in a general manner and never about how ...
GaelC's user avatar
  • 466
1 vote
Accepted

What is the best way from remove background signal from a band when doing Western blot image analysis?

I haven't done immunoblots for years, and didn't do much quantitative analysis of them even then, so caveat emptor. However: 1) "Are there any ways to determine how wide you want the band for ...
Maximilian Press's user avatar
1 vote

Flow cytometric counting of apoptotic adhering cells

Here some resources, Cell harvesting effects on Annexin V staining. Protocol for apoptotic analysis using Annexin V. Quite a long discussion about this issue. Another protocol. Cheers, Pedro
TumbiSapichu's user avatar
1 vote
Accepted

Where is DNA during the process of RNA extraction using Trisure?

Expectedly, they don’t really want to tell you what TRIsure is. What they do say is that DNA is found in the organic or interphase after extraction whereas RNA partitions to the aqueous phase. This is ...
canadianer's user avatar
  • 17.7k
1 vote

Genomic DNA resuspension after ethanol precipitation

Just a thought, have you tried eluting the DNA in Molecular Grade Water instead of TE? For DNA which are supposed to be used in downstream applications like sequencing or transformation, elution ...
Grb's user avatar
  • 61
1 vote

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

We have used Brother P-Touch printers in the stock at work, but our lowest temperature is -80C. It's ideal for making sure the labels go all the way around the vials.
Victor's user avatar
  • 11

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