23
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 experimenter to observe the behavior of the liquid and act accordingly. It is quite easy to determine when a liquid is leaking (e.g. ethanol) or when it is very ...
23
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 can handle a wide range of viscosities if you set a conservative (slow) speed for aspiration/dispense, but for solutions that don't require this, we'd like to be ...
10
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 liquid reservoir.
Pipettes can also behave rather weird with any liquid that differs significantly in any physical property from water, e.g. very viscous ...
9
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 ionic strength implies higher electrical resistance and consequently a higher electric field, provoking the faster movement of the proteins and of every other ...
8
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 nicely.
However, there is a printer available on the market (though I haven't tested it yet) which claims that they can print on any tube. This printer is called ...
7
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 biological samples with fluorescence microscopy.
There are significant challenges to achieve high-resolution cryoFM imaging.
Fluorophore characteristics at low ...
7
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 instructions to cover complex (and often bespoke) processes.
As Dan Bryant has explained, robotic systems absolutely need the additional detail in order to complete ...
6
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 appropriate
buffer in liquid nitrogen and then store them at -80°C. I would not freeze the dry pellet, as these are often hard to re-dissolve after freezing.
...
6
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 on the size of the protein(s) you're looking for.
When you load your samples in the wells at the top of the gel, then start the current, not all of the sample ...
6
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 that you need is the bead count at each point of your calibrant curve.
However, using calibration beads of a given size assumes that your cells will remain ...
5
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 with the cationic histones.(Dextran is a polymer of glucose. In dextran sulphate it is derivatised with sulphonate groups creating a polyanionic material.)
...
5
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 what you need to do if you are going to use a different rotor, but they can also be used to solve your question.
Because you are looking at a situation where the ...
5
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). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a ...
5
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 mixer, but possibly by careful, repeated inversion. The aqueous phase and the organic phase are barely miscible. The tube is centrifuged to speed up the ...
5
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 similar engines, however.
Some journals permit methods papers, but not a high proportion (and it sounds like you're looking for a place to find them, not to send ...
4
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 electrocompetent cells are more efficient than chemically competent cells. Alternatively, as you have read, you could transform your first plasmid and select for it, ...
4
Thats basically the oldest method to induce such mutations. For this purpose either radiation (x-rays) or mutagenic chemicals (like Ethylnitrosourea) have been used for this purpose. This method is undirected, so you never know what the outcome will be untill you see the off-springs. Using this method to get specific mutations is relative difficult. Its used ...
4
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 always do 2000 rpm, 20 minutes, 4C if I'm doing a midi or maxi sized prep and harvesting bacteria in a 50ml conical.
If I'm doing a miniprep I will take the 1....
4
I've run a test following this protocol to the letter...
PCR step by step:
Collect all ingredients (excluding TAQ) from the refrigerator and keep cool (on ice). Leave the TAQ in the freezer until required.
Make master mix by adding all ingredients (excluding TAQ) using fresh pipette tips for each ingredient and using the volume specified
in the PCR ...
4
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 beetles, moths, butterflies) we use a lot of visually scored data, e.g. body size, wing size, wing morphology, eye colours, bristle numbers, genital morphology,...
4
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 (mainly hemoglobin from RBCs) needs to be gotten rid of at some point. By narrowing down your input to just include cells that have DNA, you lessen the amount ...
4
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. I'm not an electrician, but higher conductivity may be the opposite of what you want. The lithium boric acid buffer claims to have less conductivity than a ...
4
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 powerful enzyme inhibitor, so if you want to apply
enzymatic steps downstream, TAE is the better choice
TAE gives a better resolution for large fragments
TBE ...
4
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 the base of the buffer and is used to set pH. Along with Tris one can use Boric acid, Acetic acid or phosphoric acid for adjusting the pH.
The buffering range ...
4
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 the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and ...
4
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 normal view! Then, go to home tab, select all the cells on the page. Go to the right side of the home tab, hit Format, then Row Height. This lets you set height in ...
4
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 directions for sample preparation because it may depend on the exact equipment.
These devices use a spectrophotometer to measure saturation, taking advantage of ...
4
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/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/phenol-chloroform-extraction.html
You can store it in -20 degrees for several ...
3
the FDA has to approve diagnostics - a rigorous and expensive process. Its a legal distinction, but also one which conveys a significant level of usefulness and reliability.
3
If you have an antibody that is directed against, for example, a bacterial surface protein, then by mixing the bacterial cells with the antibody at a suitable stoichiometry you could observe clumping of the cells as the antibody molecules essentially cross link the cells together. This would be an example of using an agglutination (clumping) assay with a ...
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