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18

The reason for degassing your gels is to remove oxygen. Oxygen in the gel interferes with polymerisation, slowing it down and making it less consistent, so degassing makes it faster and more uniform. From the EncorBio SDS-PAGE protocol: Polymerization is quicker and more uniform if you degas the first three solutions for a few minutes in an Ehrlenmeyer ...


14

The role of the salt is to neutralize the charge of the DNA's sugar phosphate backbone. This makes the DNA less hydrophilic (less soluble in water). Ethanol has a lower dielectric constant than water so it's used to promote ionic bonds between the Na+ (from the salt) and the PO3- (from the DNA backbone) causing the DNA to precipitate.


12

The only difference between FPLC and HPLC is the amount of pressure the pumps apply to the column. FPLC columns have a maximum pressure of about of 3-4 MPa, whereas HPLC columns can withstand or require much higher pressures. As a general rule, HPLC columns won't work with old FPLC equipment; FPLC columns can go on HPLCs as long as the pressure can be ...


11

Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to more tubes with 50ul max. The problem with big volumes is to get a fast and homogeneous heating and cooling. If the outside of the tube is faster, the reaction ...


11

(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki fragments). Here is a quotation taken from For the Love of Enzymes: The Odyssey of a Biochemist by Arthur Kornberg   Reiji’s research style is not readily ...


10

In my experience, shaking and mixing have different "dead spaces". Supposed you had an eppendorf tube and you stirred it around with a pipet tip for thirty minutes. You would have great convective mixing in the radial direction but virtually no mixing in the Z-direction. Supposed you had a very viscous fluid like PEG. If you set the tube on a shaker for an ...


9

what specific effect the vortexing has that makes it better than manual shaking In addition to @bobthejoe's answer about viscous fluids, (manual) mechanical shaking is also less consistent and more tiresome than vortexing. If the vortexer is always at the same speed and each sample is vortexed for the same amount of time, then the shaking step will be ...


9

You are looking for a bioluminescence imaging device. These have very sensitive CCD camera and exposure times are around 5 minutes in complete darkness.


9

You can clean up phenol by washing with choloroform, and then doing an isopropanol precipitation followed by a 75% EtOH wash (let me know if you'd like an exact protocol). To avoid contamination (and sample loss), you have to be meticulous in your pipetting (which you'll get better with practice). You can always use those phase-lock tubes which basically ...


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

How often you should calibrate your pipettes depends on the tolerances of your application. For some applications, like quantitative PCR setup, one may care a great deal; for general lab work, one can probably be less particular. You can get a sense of how accurate your pipettes are by pipetting DI water onto the platform of a sensitive (and calibrated!) ...


7

In my opinion wearing gloves during cell culture is a good idea. It works both ways as a protection: For your cells since you are not loosing small danders from your hand and for you since you are not getting media or chemicals used in cell culture on your skin when you are uncautious. I usually prefer nitrile gloves over latex, since nitrile gloves are ...


6

Assuming that you are talking about E. coli: As long as you are resuspending the cells in a suitable liquid, e.g. fresh medium or buffer, then from my experience you don't have much to worry about - the cells are very robust. I've found that different strains and growth conditions give pellets with very different qualities - some will resuspend quite well ...


6

You can run your DNA sample on agarose gel to see, whether you have significant degradation. If you are interested in contamination, you can make a standard photometric analysis to assess the 260/280 and 260/230 ratios and absorbance at 320 nm on NanoDrop or even something similar to Eppendorf's BioPhotometer. In case RNA may be an obstacle for some ...


5

Try dissolving at 50C. In the Qiagen gel extraction kit, it says to dissolve at 50C or until completely dissolved. The size of the gel is important too. Make sure it is 1.5% or less, and the size of the gel needs to be less than 400 mg. Also, pay attention to the first step. If the liquid is not the same color as the color of the first QG buffer then ...


5

When I optimized gel extraction in my hands, two factors turned out to be critical in maximizing gel extraction yield: The pH of the eluting solution. I used to elute the final product with DEPC-treated water initially, but the yield would vary a lot as the pH of the water changed. Using buffer EB is better, since it maintains a stable pH 8. Most ...


5

I've found that extracted RNA using commercial kits has stayed stable for many years at -80 C. I would certainly aliquot it before freezing however as RNA is particularly sensitive to freeze-thaw cleavage.


5

An ethanol precipitation should work. But I have had great success using the Qiagen RNA cleanup columns, which are in my opinion easier. Here is a URL to see the RNA cleanup columns Qiagen offers: http://www.qiagen.com/products/rnacleanupconcentration/default.aspx Also in the future you should consider using PhaseLock tubes: ...


5

DISCLAIMER: I am a computational biologist and haven't touched a Petri dish since I was an undergraduate biology student. I would expect that the best thing to do would be to place the lid face down. When placed so on the bench, it is only the tip of the lid that comes into contact with your (presumably sterile-ish) bench. If you place it face up, the ...


5

I've only used it for mammalian work, but the Cedex HiRes Analyzer from Roche is pretty sweet. From their docs you can analyze particles from 1-90 μm in size (the cells I work with are about 20 μm), and there are a ton of configuration options depending on your needs. As long as your cells can be stained with Trypan Blue (it's been a long time since I've ...


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

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.


4

I have been running gels with different Acrylamide/Bisacrylamide ratios recently. People usually work with 1:37.5, 1:29 ratios which are commonly used for DNA and Protein gels. I have noticed that when you work with lower ratios 1:200 - 1:500, degassing becomes fundamental to guarantee reproducible resolution of my proteins. If I don't degass the mix in one ...


4

You'll also want to make sure that you are imaging in complete darkness because you may have very low photon emission from your luciferase system and any amount of background light can overwhelm your signal with noise.


4

First thing I'd do is replace the HEPA filter. A copper plate/foil may help, and it certainly won't disrupt the flow of heat, as copper is incredibly conductive (that's why they make electrical wires out of it), and poking holes would help with the airflow, but a new filter will probably make the most difference. I've never used a full-copper incubator, and ...


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 ...


4

RNAses are enzymes, and there are various ways to inactivate them. Unfortunately, RNAses are rather stable proteins and autoclaving doesn't completely kill their activity. The common methods to inactivate are unspecific methods that will destroy any enzymes, either through covalent modification or degradation. The most common methods for RNAse inactivation ...


4

Working with petri plates both microbially and in tissue culture, there is one rule of thumb to follow. Contamination falls. Your main enemy is time, how long the medium is exposed (bar actually touching the surfaces). Ethanol DOES NOT sterilize the working surface. Think of it as a convenient scrubbant which evaporates quickly. The flame DOES NOT work to ...


4

I still prefer the good old[tm] Labnotebook. No dependencies on (proprietary) software or a computer. I generate quite some digital data (blots, sequencing etc.) this is stored on the servers of my university. We looked into this in our lab, but discarded the idea pretty fast. Most solutions are either expensive and/or proprietary. Then a lot of these ...


4

What I would usually do is transform E. coli with the plasmid, grow an overnight culture and mini prep the plasmid in the morning. Before you do the miniprep save a glycerol stock. Thus, you would have some plasmid to work with in the next couple of weeks, and also a glycerol stock to come back to. You can also keep the plate with your transformants, as you ...



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