Hot answers tagged lab-techniques
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 ...
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 ...
10
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.
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
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 ...
8
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 ...
8
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 ...
6
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!) ...
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
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 ...
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
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:
...
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 ...
3
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
Here are some possible reasons:
Your yield is very low, which you can infer from a low 260 absorbance. Either make sure your restriction digest is efficient or add more substrate to the digest reaction.
Carbohydrate contamination, which will cause high 230 absorbance. Wash the column one more time with buffer QG (the step is described in the Qiagen gel ...
3
Not really an answer I know, but too long for the comments...
This is still too broad a question. a well sealed, sterile plastic usually has a use by date, but can probably be used more than a year after you receive it. But these cases are not usually the issue.
Its sterile medium and chemicals, each of which needs to have their own due date. LB can ...
3
My personal favourite is OpenWetWare. Think wikipedia for scientific protocols and an open access lab notebook.
There's a problem with this things. Despite the common stereotype of scientist being open and good at sharing, my experience is the opposite. Many laboratories are not good at all in sharing their techniques/secrets. They will share the basic ...
3
There's Benchfly, which is a video-based protocol library:
http://www.benchfly.com/video-protocols.php
There's also JOVE, which is a peer-reviewed video journal that sometimes covers protocols:
http://www.jove.com/
3
Beta-galactosidase (B-gal for short) is an enzyme that will process the substrate lactose. In applications using B-gal as a reporter (lacZ gene), two lactose analogues are commonly used: X-gal or ONPG (as pointed out by @Alan Boyd). Both substrates are colourless, becoming colourless once hydrolyzed by B-gal. The formation of this colour allows B-gal to be ...
2
When doing a recent investigation into methods of quantitatively determining the concentration of aspirin in solution I found these procedures helpful. They are a rough guide; design and procedure will vary by model. It may be wise to check the manual that came with it, however in summary:
Calibration
Performed at least once per week
Performed in a room ...
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
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
Immunopanning is essentially an immunoprecipitation (IP) of cells using an antibody immobilized to a solid surface, like a cell culture plate. Conventionally, an IP is performed using small agarose or magnetic beads (~50 to 150μm in size) conjugated to an antibody or Protein A/G, and can pull down individual proteins, protein complexes, and/or nucleic acid ...
2
I asked my professor, and the answer appears to be differences in both the generation and the final product.
Free chromosome fragments are created through irradiation/other damage of the germline in one animal. Through a series of crosses, it is possible to introduce individual fragments (containing a duplication of your gene of interest, as well as a ...
2
Reverse transcriptase (and most other commercial enzymes these days) is made recombinantly, then purified to varying degrees, using varying methods, among different suppliers (and sometimes between different lots from the same supplier). Some enzymes actually have multiple functional units combined in a complex, others may require a post-translation ...
2
This may be what you are looking for. The conversion formula is:
$$
\text{Protein concentration (mg/ml)} = \frac{ (\text{Absorbance at 205 nm})} {31}
$$
The first, Scopes, does indicate that nucleic acid contaminants will confounding 205 nm absorbance readings. It seems as though this spetrophotometic calculation is feasible for relatively pure protein ...
2
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 ...
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
If you use pre-staining, the stain migrates as well in the electric field and this can lead to some parts of the gel being unstained. This would look pretty much like you describe, the ladder being invisible as well points strongly to that part of the gel just being no stained properly.
It sounds a bit unlikely with your running parameters, I've only seen ...
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