Capillary electrophoresis versus slab gels

Slab gels and capillary electrophoresis both pull charged molecules through a matrix. They do not produce the same record, and the protocol you want is the one that produces the record the decision needs. A slab gel, agarose or polyacrylamide, ends as a photograph. A capillary system ends as an electropherogram, the trace used for fragment analysis and for Sanger sequencing. The photograph at the top of this page is the slab case: an agarose gel with glowing bands under an orange safety shield. How to read that photograph is agarose gel electrophoresis for DNA. This page is the choice between keeping that kind of picture and sending the sample to a capillary.

Stage one: name the record

If the next action is "do I see a band near this size, in all these lanes, and can I cut it", stay on a slab. A molecular weight ladder in a neighbouring lane is the scale. Twelve students or twelve clones fit on one image. The failure is visible as a smile, a smear, or an empty well.

If the next action is "what base is at this position" or "do these fragments differ by a small length I must tabulate", the capillary trace is the record. Fragment analysis reports peaks sized against a standard that co-migrates inside the same capillary. Sanger reports a sequence trace from a primer. One read does not cover a long insert. Mixed peaks mean mixed template. Identity of a clone is the chromatogram plus the map, not a colony PCR band alone. The public sequence you align against can be checked in the NCBI Nucleotide database. The alignment does not create the trace.

A western blot is a third record. It is a slab protein gel transferred to a membrane and probed, as described in western blot from gel to membrane. It is not an electropherogram, and a capillary DNA trace is not a blot. Protein capillary methods exist as their own instrument class. The choice most benches face is still the photograph versus the DNA trace.

Stage two: resolution and the matrix

Agarose slabs resolve ordinary PCR products, plasmids, and digests on a scale of tens to hundreds of bases, depending on percentage. Two bands that differ by a handful of bases usually will not separate. Polyacrylamide slabs tighten that window for small DNA and are the usual matrix for proteins. You still leave with a picture and a lane ladder.

A capillary filled with a replaceable polymer, run at high voltage with detection at a window, can separate fragments that an agarose photo stacks together. The polymer, the capillary length, and the internal size standard set how small a difference you may call. There is no universal "one base" guarantee on this page. Read the instrument's size range. Outside that range, peaks bunch or drop out, and the call is not better than a careful gel. It is only harder to see that you have left the window.

Denaturing conditions are part of many capillary DNA methods, so the length, not the shape, dominates. A slab of native agarose still lets shape move plasmids. Do not compare a supercoiled band on a photo with a denatured peak size and expect one number.

Stage three: the sample each format will accept

A slab well takes a small volume made dense with loading dye. Particulates usually just look ugly. Salt makes the band frown or smear, and you can still see that on the image.

A capillary injects by voltage or pressure from a much cleaner sample. Particles clog the end. Air introduced with a sloppy fill stops the current. The sample buffer, often a formamide-containing loading solution for denaturing fragment work, is the one the instrument method names. An internal standard is mixed in, which is a different idea from a ladder in the next lane. Too much salt, or leftover ethanol from a cleanup, changes injection and shifts or weakens peaks. The amount and the cleanliness you should confirm before a sequencing run are set out in what to check before a sequencing run. Those checks apply before either a Sanger capillary or a larger sequencing workflow. They are not a gel photo of the PCR alone.

Stage four: failures that belong to the hardware

Air in the capillary is an open circuit. Current fails, the trace is empty, and the samples may be recoverable if you have not baked them in a bad injection. A clog from dried polymer, dust, or a precipitate looks similar, sometimes with a pressure error when the instrument tries to refill. Polymer that is old, or that was stored warm against its label, fills with microbubbles and gives noisy current and wandering mobility. Replace the polymer and confirm a size standard before you reinterpret alleles.

A blocked capillary is not a null genotype. Rerun the standard. If the standard's peaks are missing or spaced wrongly, the biology is not yet the question.

Slab failures are the ones the photograph shows. Wells that leaked, a ladder that did not open, a field reversed so the bands are gone, a stain and lamp that do not match. Those are repaired by a new gel. They do not become a base call if you stare harder. Ultraviolet documentation of that gel stays behind the orange shield.

When to stop and change format

Stop cutting bands out of agarose when the two species you must separate never part on any honest percentage. A capillary size, or a polyacrylamide slab, is the next format, not a longer UV exposure. Stop sending samples to a capillary when all you needed was "the PCR worked" for twenty clones. The photograph is the lighter record for that question, and the shield-and-stain hazards are the ones you already know. Stop treating a noisy trace as a variant call when the size standard in that injection is itself distorted. The standard is the control. A distorted standard withdraws the sample peaks.

Next-generation runs are a further format, with libraries and indexes, described in next-generation sequencing from library to reads. They are not a longer capillary. Choosing them is a different decision from the one on this page.

Safety and the room the instrument sits in

Slab hazards are the power supply, the stain, and the UV box. Capillary hazards add a high-voltage instrument, polymer chemistry from its own sheet, and often formamide in the sample mix, which is handled as the chemical hazard that sheet describes. Neither format is a diagnostic licence. Biosafety follows the organism the DNA came from. The WHO context for that judgement is institutional. This article does not make it.

Polymer and formamide-containing mixes do not enjoy a hot bench. A vial left warm can bubble or drift. After a power cut, an injection that was mid-run is a partial trace. Rerun it. Do not average the missing minutes into a base call. In a building where the line sags, an instrument that reports unstable current wants a standard injection before the sample plate is trusted.

What an enquiry should carry

EVRINTH can take a sourcing question. State whether you need a slab photograph or an electropherogram, the size difference you must resolve, the sample matrix, and whether the downstream step is a cut band, a fragment table, or a Sanger read. The molecular biology catalogue is where general reagents sit, and the quote request is where the specification goes. The nucleic acid analysis pathway frames the gel or the trace inside a longer workflow. Ask whether a quotation is possible. A request that says only "electrophoresis" will come back as a question about which record you meant.