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13

The answer to the first part of your question can be found on Wikipedia: Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modified with acidic side-groups, such as sulfate and pyruvate. The neutral charge ...


5

Yes, you can do this. As long as the final mix has the proper concentrations of everything, its fine. Just make sure you compensate the by adding 30 uL (the extra volume) less of water to the master mix.


5

The cited article describes effects of Tris on the outer membrane of E. coli. Like all Gram negatives E. coli has an inner (or cytoplasmic) membrane which is a typical phospholipid bilayer membrane containing many membrane proteins including transporters and, of course, an electron transport chain. Surrounding this there is a layer of peptidoglycan, and then ...


4

It's hard to know what RNaseZap does since the ingredients list is a trade secret. However, I expect it is a lot more than just detergent. RNaseA is extremely hard to destroy; moreover it can easily renature once the denaturant is removed. Therefore, very minute quantities are sufficient to annihilate RNA experiments. The historical method of purification ...


3

Storage conditions and shelf life are some of the things that you should consider in comparing columns.


3

Extracellular matrix (ECM) fluoresces, especially Collagen and Laminin. The maximum is in the DAPI and FITC channels and the fluorescence becomes weaker towards longer wavelengths. However, since the coat on the TC flasks is very thin, I would not expect this to be a problem. The best thing is just to try it. There is also a quite famous document available ...


2

SYBR green is designed to be much less carcinogenic than ethidium bromide (EtBr). All these gel dyes work by intercalating themselves into the DNA stack between the bases specifically which has a great potential for causing mutations and messing with the workings of the nucleus. My remembrance is that the SYBR and GelGreen/Red etc dyes are large and ...


2

Regardless of what protocol you use, and what the advertised efficacy of that protocol might be, in any situation like this I think the important thing to consider is: what would happen if the material taken from a re-used column was contaminated by a previous application? Can you live with the consequences of such contamination? If you are preparing DNA ...


2

Seems like you have covered the essentials here. I can't think of anything else. Since the Qiagen patent on spin prep columns ran out, these kits are very cheap - $0.40 each? In the 3 or 4 kits Ive used, they all seem to use the same protocol and about the same buffers, so there might be differences in quality or yield but if so, they are small. You ...


2

Working with hot phenolic solutions is rarely been done due to the dangers of this. Phenol needs to be handled in the fume hood anyway, at higher temperatures it gets even more volatile. So if you plan to do this, do it with extra care to avoid injury. There is one paper that describes this method for bacteria, but this should work for other samples as ...


2

To increase storage life: after gel solidifies, dampen it with running buffer. Wrap the gel in polyvinyl chloride. Place in plastic container with a lid. Store in fridge in dark. It will last for a year as long as you re-dampen it with buffer each time you access it. Don't keep it submerged in buffer as the etbr will diffuse out. this is probably the cause ...


2

I think I have some evidence that the key factor is light. Since asking this question, I changed the buffer for fresh TAE-EtBr (same concentration as in my gels) moved my gels from a well-lit area into a closed, opaque box so that they remain in darkness. After 1 week, I ran equal amounts of my ladder in the stored gel, as well as a fresh gel. The results ...


2

I always add the Taq to the mastermix. First it makes the handling easier and it avoids pipetting steps which can cause contamination and can also be forgot. Then the enzyme is very stable and will even tolerate room temperature without problem. Since we are going to heat this 30-40 up to 95°C, so this is clearly not problematic. Since the reaction mix is ...


2

Good question. I found this reference in "Molecular Biology: A Project Approach" A phenomenon called photobleaching occurs when ethidium bromide (EtBr) -stained DNA is illuminated by ultraviolet light.... This decreased fluorescence is presumably due to the dissociation of ethidium bromide from the DNA. Ethidium bromide fluoresces when it is in a ...


2

Autoclaving is the best way for sterilization. If your growth medium is large, how much chemical disinfectant do you think you would need to bring up to its working concentration? Add some acid to your waste medium (so that it's only weakly acidic), not as disinfectant, but to suppress ammonia formation. Urea will still break down during autoclave, but ...


1

Trypsin is a protease, an enzyme that cuts other proteins. It is used in cell culture to help lift cells from a plate by cutting the proteins that hold the cells to the plate and to each other. Trypsin can go bad because a trypsin enzyme can cut other trypsins, eventually destroying the batch's enzyme activity. This is why trypsin must be kept frozen. Of ...


1

I use virkon for cell cultures, but for viruses I tended to use standard bleach during the time I worked in a virology lab.


1

The Ambion's RNAseZAP is not a detergent but contains chemicals that denature RNAse, the step of rinsing with water is necessary in order to clean up the chemicals as well as the denatured proteins left. It is highly recommended as it completely wipes out the RNAse and the experiment can be run safely. No need to risk time, money and samples. So, to sum up, ...


1

Using DMSO (dimethyl sulfoxide), agarose can be separated. After heating and stirring around 2 hrs you will get a yellow stiff gel of agarose by filtering.



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