Electronic form for SDS-PAGE - Software developer needs help (Aug/22/2006 )
A company Im working with has a NIST grant for developing data gathering systems for labs. The lab that they were working with is now "too busy" to provide any assistance in completing the form for data gathering for SDS-PAGE experiments.
As a software developer with no bio or lab background and no support from the lab anymore its pretty much impossible for us to complete this form. If anybody could provide some assistance I could post pictures of what we have and try to ask some questions that Im sure you'd find humorous.
Thanks.
Sounds interesting... Can you give us some more details?
Sure, where do you want to start
We have a XForms based data collection system that stores data in an XML database back end. The forms are created using an XHTML like language that provides the display for data collection.
In this case I have a conversion of a crude paper form that the lab was using to record info on a series of SDS-PAGE experiments. The first page looks like the info about the make-up of the gels and what goes in what lanes.
The 2nd page, which is a work in progress for us on the technical side, is where an image of the gel would be placed.
An image of the first page is attached.
I'm not sure what it is exactly you're after, but I'll try to help (I'm a programmer and a molecular biologist)...
The top of the form is for basic gel identification (title, purpose, operator, gel number). The gel number should likely be auto-assigned to avoid duplication, and be unalterable (like a ledger entry or PO number in an accounting system). You could make it semi-descriptive, perhaps based on the date of the run. BTW, you need a date field, and perhaps a field to cross-reference the gel to a particular (paper) lab notebook and a page number.
Then we start to get into experimental conditions.
I'm not sure what "type" is; it could mean dimensions of the gel (as I see thickness is recorded, by not height and width). It could also mean "native" or "denaturing", meaning essentially whether the gel had SDS in it or not (but I see the SDS information is elicited elsewhere -- the denaturing agent might be urea as well). It could also refer to the type of sample being separated -- the gel might be a DNA gel, a protein gel, a polysaccharide gel, an RNA gel, etc. Or perhaps it intends to indicate subsequent use of the gel -- the gel might be intended for Comassie or other staining, or it might be intended for use in a Western or other blotting procedure. Or it could be seeking whether the gel was of a uniform percentage or a gradient gel, or what the buffer/ion composition was (Tris-Glycine, Tris-Tricine, Tris-Acetate, Bis-Tris, etc.). Finally, I guess it could be looking for one-dimensional or two-dimensional, discontinuous or continuous, etc...
So, I'm really not sure what this field expects; this surely indicates that the label "type" is insufficient.
The "resolving gel" and "stacking gel" entry areas should really be reversed, because on the actual gel the stacking gel sits atop the resolving gel, so by having these in opposite orientation on the entry form you're just begging for data entry errors 
. The stacking gel's purpose is to concentrate the sample before it enters the resolving gel. The stacking gel is of a different composition (see below) than the resolving gel, but they are two sections of the same "gel" -- this is the genesis of the term discontinuous SDS-PAGE (continuous SDS-PAGE would not use a stacking gel; this is rarely used). This also means that it makes no sense when you, later on, allow for the possibility of the stacking gel being purchased from one company and the resolving gel from another.
I'm similarly confused by the buttons labeled Bis and Acr. The abbreviations are clear enough, but all gels that I'm aware of contain both acrylamide (Acr) and bis-acrylamide (Bis, also known as N,N'-methylenebisacrylamide). Gels are described as percent solutions (w/v), and their resolving power is determined by their pore size. Pore size is determined by the total percentage of acrylamide (called %T, arrived at by adding the percent acrylamide and the percent bis-acrylamide) and the percent cross linker known as %C (bis-acrylamide is the cross linker; this value indicated what percentage of total acrylymide is cross linker). Confused yet? 
As an example, a gel prepared by dissolving 30 grams of acrylamide and 0.8 grams of bis-acrylamide in 100 ml of volume would be 30.8 %T (((30 g + 0.8 g)/ 100 ml) x 100), 2.6 %C (0.8 g/30.8 g x 100). So, I'm not sure the purpose these radio buttons serve. These buttons imply an either/or situation, when it is not so. A gel is adequately described by its size (height x width x thickness), the composition of its buffer, and the %T and %C of the stacking and resolving gels. In pratice, %T and %C are left off in favor of saying the gel was, for example, "a 12-well, 8 x 10 x 1 cm, 4% - 12% gradient Tris-Tricine mini gel purchased from Invitrogen". However, true accuracy and reproducibility require knowledge of %T and %C, dimensions of the gels, and the buffer used. Also useful is the electrical properties of the run and the temperature of the run.
Gels can be run at constant voltage (V), allowing the current to fluctuate, or at constant current (mA), allowing the voltage to fluctuate. Most gels are run at constant current, as this allows better temperature control (if the current goes too high, too much heat will be produced, and the gel will be distorted or melt). Gels are typically run at room temperature or in the cold room (4°C), but some will have temperature controlled buffer recirculated throughout the run, thus totally controlling temperature.
Is this the kind of thing you're looking for?
How is this done in the "real world"? Are the gels inventoried when the come in an assigned a number? Or does each lab do it in some sort of ledger? If the latter, what info is recorded along with that?
As to things being unalterable, we are working on figuring out the "rules" that would be used that would allow for modifications caused by data entry error etc.
As to notebook page numbers, the app is supposed to supplant all or part of a paper notebook, but what type of particulars besides this data would need to be recorded for it to get, lets say 90% coverage?
Neither was I - it was just copied off the paper form where they had multiple check boxes. The drop down has "Laemmili", "Small Pore" and "Other". Guess its probably junk eh? May have been too specific for the work doing done by the tech that set up the paper form.
This also means that it makes no sense when you, later on, allow for the possibility of the stacking gel being purchased from one company and the resolving gel from another.
Some of the feedback from the lab before they became busy was to have most of the gel data auto populated via prepared products. So what I think we probably need here is a pair of drop downs with a manufacture and a product selection, with the cop-out of "Other". Does that sound sane?
Actually things are getting much better. Again, the Bis, Acr were copied from the paper form. They didn't give any explanation to us. It seems like the %T and %C are much more important for future data mining. I take it that these values would be stated by pre-made products.
Oh, you talk about "wells" here, but we have "lanes". Important? We also have 10 - what are typical/max values? Also, what exactly is the physical layout of the A and B lanes? Do they alternate, or As first Bs next? The 2nd page will have the image of the gel placed on it and we want to transfer over the labels from the first page.
So sounds like just recording the physical characteristics of the gel. We dont have anything for the "buffer" do we? What type of info goes in there and is it determined by the manufacturer of the gel?
Pure gold. Thanks alot. We'll start making changes to page 1. Then there is page 2. I'll get to work on both, but first off to the dentist...
In my lab, the blank gels themselves are unimportant; there is no identifier assigned to them. We buy pre-cast gels in boxes of ten, but many people cast their own. After a gel is run, it becomes important. I'm not sure it needs to be assigned a number; my suggestion came from the fact that you had such a field present. What we do in my lab (if the gel has been stained) is either scan the gel and save the image as a .jpg file, or dry the gel between two pieces of cellophane and tape the dried gel into a lab notebook.
It may supplant the need to record the characteristics and results of a gel in a notebook (paper or electronic), but there are other data concerning the acquisition of the samples loaded on the gel: the source of the samples, the conditions under which the source of the samples were grown, the method used to recover the samples from the source, etc. -- that would likely be recorded elsewhere.
The gel's image is the endpoint of experiments leading up to the running of the gel, to keep the gel in context, it seems it would be necessary to maintain some kind of cross-reference to that prior data. It might also be nice to maintain some linkage to prior gels in the same experimental endeavor -- as shocking as it may seem, frequently things don't work the first (or the tenth
) time; I frequently find myself adjusting conditions and then refering in my notes back to a prior gel ("Changing condition X seems to have helped tremendously, compare lane 3 above to lane 6 on the gel run on 7/28/06; it's apparent that changing condition X from A to B helped considerably...").As you can see from my prior post, there are many different types of gels. Some information regarding the gel type should be recorded, I'm just not sure how deep you need to drill down here.
A "Laemmili" gel (BTW, it's actually spelled "Laemmli") is another way of saying a discontinuous gel (i.e. one composed of a stacking gel and a resolving gel); the name comes from the author of the paper that first described such a system (Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680-685).
Autopopulating fields based on the type of gel purchased would be a great time saver, and minimize data entry errors. What language is the interface written in? I would include several branch points in the code, if possible -- if one answer negates the validity or neccesity of another field, why even show the second field? If this were web-based, I'd be looking at AJAX.
Well, one would hope so, but frequently these data aren't provided, or at least not completely. Using Invitrogen as an example, the data on their gels is here. The composition of the gels is described (see especially the data on page 6 and page 9), but not completely. I'm not sure whether they consider this propritary, or whether they'd give up %T and %C with a phone call. In reality, most people (in published works anyway), just describe the percentage gel used, its size, and the manufacturer.
Wells are the voids into which you load your sample, lanes are the result of running the gel (a sample is loaded into a well and sepearted into a series of vertically stacked bands in a lane). I was a little confused by the second page -- the boxes labeled 1 - 10 I would expect to correspond to wells (and thus lanes) of the gel, I was confused by the further characterization of the input into boxes labled "Gel Lane A" and "Gel Lane B".
The only thing I can think of is that most SDS-PAGE electrophoresis units allow two mini-gels to be run simultaneously, thus the form may be referring to Gel A and Gel B (and "Lane" was a typo) run at the same time in the same unit. But this is not as helpful as it may seem; for unless the two gels are duplicates of one another, it's possible that the data solicited above this section will only apply to one of the gels.
As to number of wells (or lanes) on the gel, there are many possibilities. The most common gels used have 10 or 12 wells, but I have in my lab now some 15 well gels and some 1 well (preparative) gels. This is an area where it would be nice to have page 2 react to data entered in page 1 -- if you solict the numer of wells in the desription of the gel, you could have page 2 provide the appropriate number of sample data input boxes.
I may have been a bit unclear here. Discontinuous SDS-PAGE is considered discontinuous not only due to the presence of a stacking gel, but also because it uses a discontinuous buffer system: different buffers are used to prepare the stacking gel, to prepare the resolving gel, and to run the gel.
SDS-PAGE utilizes three ions: the so-called leading ion, the trailing ion, and the common ion. Together, these determine the electrical properties of the gel. These ions are determined by the buffer used to cast the gel (what the acrylamide was dissolved in) and the running buffer(s) used in the electrophoreisis unit. Thus a Tris-tricine gel run in MOPS buffer would behave differently than, say, a Tris-acetate gel run in MES buffer. So, you should solicit the gel type (e.g. Tris-tricine, Tris-acetate, etc.) and the running buffer used (e.g. MOPS, MES, Tris Glycine, etc.). The pH of the buffers used is also important -- so a stacking gel may be made with Tris-Cl pH 6.8, while the resolving gel might be made with Tris-Cl pH 8.8, and the whole thing run in a Tris-Glycine running buffer at between pH 8 and 9.
The physics of all this is quite complex, which is why most people just publish that they used a "12% mini gel from Invitrogen". There's a pretty good overview of this buffer and ion stuff here and at other sites on the web.
Correct on all counts. The time field could be minutes or hours, depending on the size of the gel.
Ouch. Hope it's just a check-up with no pain involved...