DNA Marker – λ (Lambda) Ladder
These are basic DNA sizing markers for use when checking other DNA samples on an agarose gel containing a DNA stain, such as Ethidium Bromide (EtBr).
Two different double restriction digests of Lambda Phage DNA produce two ladder patterns. These are used to help identify the fragment size of other DNA samples. This may help confirm validity of (among others) a PCR product, a cloning construct after digestion or just a mini / maxi prep of a particular plasmid / cosmid.
The ladder, along with DNA samples are normally mixed with a loading dye. This is advantageous for two reasons: firstly, it aids the loading of the sample on to the gel; secondly, when the gel is running you can estimate how far the fragment of a particular size may have migrated.
Xylene Cyanol runs at ~4kb, Bromophenol Blue at ~550bp and Orange G at ~200bp.
Digestion of λ HindIII Fragments
λ HindIII fragments (pre digested) are purchased (Invitrogen, 500μg @ 0.5μg/μL) and the second digestion is set up to create the ladder of choice.
To produce 1x250μL tube, enough for 50 gels at a concentration of 0.2μg/μL (1μg per 5μL) do the following digestion:
| Total volume 220μL: || || ||
| || λ HindIII fragments || 100μL (50μg)
| || Buffer (10x) || 22μL
| || EcoRI or NcoI || 3μL
| || dH2O || 95μL
Mix well and incubate @ 37°C overnight.
After incubation add an excess of 10x Loading Dye - 30μL, to give a final volume of 250μL.
Heat the digest @ 65°C for 5 minutes then transfer to ice. Check the new ladder on a gel using a previous digested ladder as a reference. Multiple digests can be setup and the tubes not in use are frozen.
Use 5μL of the λ Ladder or λJ Ladder per gel, thus loading the recommended amount of 1μg.