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9

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 ...


7

This is particularly an issue when you do maxipreps and the pellet is 200x the size of the pellet from a miniprep. The reasons why? Maybe you're pelleting at too high of a g-force (pure speculation). Alternatively, you could be growing the cells too long. I typically miniprep after a 12 hr culture. If you're seeing any darker colors in your pellet, you have ...


6

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 ...


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

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

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 ...


4

What I do to avoid this is to ressuspend the cells by pipetting up and down the P1 (ressuspension) buffer on the side of the tube. This way, every time takes only a bit of cells every time and big clumps are avoided. The only times I used to get big clumps hard to ressuspend were when I went poking at the pellets first.


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 ...


4

We routinely use E. coli preps from 16-18h overnight cultures. The cultures don't show any overt signs of lysis (sometimes you can see cellular debris if the culture has indeed overgrown). I have also noticed inconsistent times when resuspending the pellet. For my own use, centrifuging at too great a g-force or for too long can increase the likelihood of ...


4

If I recall correctly (from when I interned at ABI), you can't directly set this in the Veriti interface. However, (again, I'm not 100% certain -- I couldn't find anything to confirm this after 30 minutes of googling,) the ramp rate percent refers to the percent of maximum ramp rate for the heating block. According to the product website, the max heating ...


4

I believe the standard is an graded-speed treadmill test. An example pub: Genetic variability in forced and voluntary endurance exercise performance in seven inbred mouse strains If there is a particular ethical concern, you should always have your institutional review panel review it. If you call up your department of animal resources (or what ever 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

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

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 ...


3

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 ...


3

If the mice will be incapable of walking on a treadmill, which suggests that they will be very ill, you could just measure their homecage activity. There is some normal, baseline level of activity in a cage. You could compare controls to placebos to experimentals. If the drug you are testing increases homecage activity, then it would appear to be effective. ...


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

This is a perfectly reasonable result. Remember that you are measuring an average value for a very large population of BSA molecules. Essentially what the 0.4 means is that at any one time, about 40% of the BSA molecules have a free thiol, while the rest of the cysteine residues are disulfide-bonded.


3

The binding of proteins (and cells) to glass (or silicon) surfaces can be prevented by coating the glass with polyethylene glycol (PEG) groups. PEG-silane is a reagent used to create this coating. PEG-silane (the image shows a methoxy- version) (image taken from here; no connection) will coat glass surfaces because the silane portion (right hand end of ...


3

The paper you cite says that the break points are single stranded DNA which have specific proteins bound to them. I'm not an expert here, but if thats the cause of meitotic break points there are some interesting possibilities for detecting them: you could detect them with a tiling array. - that's an micro array which has an oligomer every 40 bp or so ...


3

As Thomas pointed out, there is a maximum ramp rate which you can set up on the thermocycler. Its value can go from 0% to 100%. I can then assume that a 100% ramp rate will corespond to the maximum ramp rate of 4.25°C/s for the heating block or 5°C/s for the sample. which means that a ramp rate of ~2.4%. Subsequently, I set up my reaction at a 2% ramp rate, ...


3

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


3

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 ...


2

Proteinase K activity is greatly increased by addition of denaturing agents like SDS or urea (Hilz et al., 2008), indicating that the denaturation of the substrates helps Proteinase K to degrade them. Increasing the temperature to 50°C will also unfold some proteins already, making it easier for the Proteinase K to degrade them. The proteinase K seems to be ...


2

In your linked wiki article: "An elevation of the reaction temperature from 37 °C to 50 - 60 °C may increase the activity (of Proteinase K) several times." The enzyme works faster at 50°C.


2

It is actually surprisingly easy and reproducible to make emulsions with defined components. We do it routinely in our lab: http://www.sciencemag.org/content/332/6026/209.full - CBT protocols http://www.sciencemag.org/content/336/6079/341.full - CST protocols



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