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 ...
- 471
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 ...
- 634
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 ...
- 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 ...
- 474
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 ...
- 8,256
5
votes
Accepted
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 ...
- 3,346
4
votes
Accepted
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 ...
- 51.5k
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/...
4
votes
Accepted
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 ...
- 44k
3
votes
Accepted
Protocol to dilute DNA step ladder?
You'll probably have to titrate it down yourself. You might be able to estimate: The detection limit for ethidium bromide staining is about $1 ng$ per band. The insert says it's at "1 $\mu g/ \mu l$", ...
- 196
2
votes
Accepted
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 ...
- 464
2
votes
Accepted
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,...
- 1,313
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 ...
- 466
2
votes
Accepted
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 ...
- 7,637
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 ...
- 226
2
votes
Accepted
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 ...
- 3,832
2
votes
Accepted
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 ...
- 11.2k
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 ...
- 9,122
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
- 982
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 ...
- 17.6k
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 ...
- 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.
- 11
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