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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 ...


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.) ...


4

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 ...


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, ...


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

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, ...


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

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


2

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 ...


2

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.


2

My guess is its the solubility properties of the anions. Chloride ions can precipitate with some metals that might appear in a complicated buffer or medium. And yes they could even compete with enzyme binding. Sulfate will remain in solution with just about anything. There are exceptions, but if you're making a buffer you're probably going to do better ...


2

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 ...


2

What you are asking about is the precipitation of DNA (or any other nucleic acid) by isopropanol (or ethanol, which is more common). To do so, you add salt (usually slightly acidic sodium acetate) which makes sure that the phosphate backbone of the DNA is saturated with sodium ions to make it less soluble. Then you add the organic solvent, which precipitates ...


1

For ligation of a linear molecule to occur, the two ends must come together at the active site of the DNA ligase. In a simple molecular cloning experiment the aim is usually to avoid recircularisation of a cut plasmid vector, and instead to get a new fragment of DNA inserted into the vector. However because the ends of the linearised circular plasmid are ...


1

I'm a HUGE fan of FASP (filter-aided sample preparation) which is an in-solution preparation/digestion. It is very fast, allows you to not worry about a protein precipitation step, and gets rid of all MS incompatible substances with the wash steps (so doesn't matter what lysis buffer you use). It requires very few, inexpensive materials (a pack of 100 ...


1

I did try another transformation: again with our electrocompetent cells Cells form other lab chemically competent cells I made The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert. On ...


1

Yes, they will both affect pH. In addition to the above comment, both, but methanol especially dehydrates samples - removal of water will most definitely affect the pH in organelles. This will be time dependent. Both duration of fixation, and time after fixation before you assess the cells will affect the pH of the organelles. Because you fix the cells in ...


1

In my opinion, cell fixation shouldn't change the pH. However unbuffered formalin will oxidize and lower the pH, but using PBS should buffer around pH 7. Maybe Glutaraldehyde fixation would also be an option, if the others are not working... I found this website by leica very useful, maybe it will also help you: ...


1

So after a lot of work in optimization, I thought I would post what worked best for me. This buffer recipe was able to successfully transfer EGFR and insulin from the same lysate, and a clear band for both (large and small protein respectively). 10% SDS-PAGE gels were transferred at 1A for 10 min. High Current Transfer Buffer 48 mM Tris 15 mM HEPPS 1.0 ...


1

New England Biolabs, which has an excellent reputation for well-tested, well-documented, and robust products, has a line of protein expression and purification products that I'd definitely take a look at. I've used the pMAL system for expression of fusion proteins in E. coli, and the PURExpress system looks just like what you're looking for. I have a ...


1

Look at chemical hydrogen bond breakers. Guanadine hydrochloride is included in the buffer conventionally to compete with the the hydrogen bonds that form the double helix. Other hydrogen bonders like Propionamide and 2-Pyrrolidone can weaken the helix and I would think lower the melting point of the bonds. How much the Tm is affected would depend on the ...


1

Perhaps isothermal amplification is possible (NASBA)? Amplification of DNA also seems possible (NASBA at biomerieux).



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