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Bob Eisenman

Photographing size fractioned DNA with UV

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@Marty Backe

Very tech...a bit out of date photographically....you might get the idea from the pictures if comprehending the text goes over your head.

Bacteria have a protective mechanism for fending off infection by virus. In a viral infection by a virus the injected viral dna takes over dna replication and reproduces itself. An evolutionary developed protective mechanism for bacteria is the production of restriction Endonuclease with a dna site specific recognition site. It cuts 'foreign dna' to smaller dysfunctional  pieces.

New England Biolabs' recognized the potential for manipulating and cloning dna. It established a product line of restriction Endonuclease and other products.

EcoR1 is a restriction Endonuclease I ordered frequently from NEB along with several other RE's.

EcoR1 restriction endonuclease

The second item bacteria were found to have is a small satellite of dna called a plasmid. The  plasmid replicates at the same rate (called copy number) as the remainder of the bacterial dna.

Pioneering genetic engineers modified the plasmid to replicate to higher copy numbers than wild type and introduced 'poly cloning sites' consisting of various pairs of restriction endonucleases.

The concept of cloning is to first cut the plasmid (example bluescript) with a specific Restriction Endonuclease and also the dna of interest (a patient dna or dna containing a gene part). Without going into detail the RE specific cut plasmid and the RE cut specific regions of interest is mixed with 'ligase' and some other chemicals which integrates the plasmid into the polycloning site.

This plasmid-cassette is reintroduced into bacteria in a process called transformation of competent cells. The bacteria is incubated overnight, the bacteria+plasmid replicates and the included plasmid-dna-cassette replicates to a high copy number. A zerox machine for dna ! The next day a procedure for recovering the plasmid-dna-cassette is carried out. A small amount if culture might be mixed with glycerol and preserved at low temperature in a freezer for later culture growths.

Recutting the plasmid-cassette with the same Restriction Endonuclease releases the cassette-dna segment of interest from the plasmid at the polycloning site. To separate the plasmid from the released cassette (dna of interest) a small amount of the mixture is applied and electrophoretically run through an agarose (from seaweed) gel for a period of time.

At the end of the gel run a small amount of (in the old days when carcinogens were not frowned upon) ethidium bromide (etBr) in water is applied to the gel in glass tray. EtBr intercalates rapidly into the DNA double helix , while still in the gel. To see where the dna is one views the stained gel under UV light (UV light box). EtBr fluoresces in the visible (wearing gloves to protect the fingers from EtBr).

UV box

In the old days before digital cameras were available the gel was photographed (at least where I worked ) with Polaroid type 54 (57?) film in a mechanical film holder above a bellows type camera using a lens and a special filter to block non specific fluorescence.

Polaroid type 57 filmFilm cassette holder for Polaroid type 57 film

 

Film cassette holder for Polaroid type 57 film

The result from the dark room camera looks like:

 

EtBr stained, size fractioned dna on an agarose gel

 

EtBr stained, size fractioned dna on an agarose gel

 

A dna ladder of incremental size is used determine the fragment size. A razor blade is used to excise the fragment from the gel (like thick jello) and perhaps send it off to the dna sequencing lab or for use as a PCR template. PCR is polymerase chain reaction occuring in a thermal cycling machine on the bench top.

 

I would go through boxes of Polaroid type 54 film (or was it type 57?) applying 'goop' to the photo as a film surface preservative and taping the photo into a daily notebook. I've taken hundreds of such photos in the 1990s. Perhaps creating two dozen lab notebooks (before digital media storage was developed).

So there you have it….Restriction Endonuclease, plasmid, polycloning site dna-cassette, EtBr, agarose gel, Polaroid type 54 (57?) Film, size fractioned dna, dna cloning

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