In the past, size markers for gel electrophoresis were self-made. Each laboratory had its special plasmid. Restricted with certain restriction endonucleases, it revealed good markers with irregular bands. Today, most researcher prefer standardized size marker with a regular banding ("ladders").
AppliChem provides ready-to-use DNA markers and lyophilized DNA markers. The latter are extremely stable, since the phenol extraction, which is part of the manufacturing process, inactivates and removes all enzymatic activity. The lyophilized markers are shipped at ambient temperature and are stable for several years!
Besides DNA markers, AppliChem offers a number of protein markers for SDS-PAGE and Immunoblotting, prestained or non-stained.
Tips on the use of DNA length markers, for more details download our brochure!
• Correct storage: Deproteinised and lyophilised DNA samples are extremely stable (> 5 years). Problems do not usually occur unless DNA markers in solution are stored (> 6 weeks) at room temperature, they become contaminated with bacteria, or are frequently thawed and refrozen (> 20 times). After dissolution of the DNA, we therefore recommend aliquoting of the DNA marker in ready-to-use aliquots for storage at -20°C (for 2-4 years). At +4°C, markers in solution are stable for several weeks or even months. Here, however, there is the risk of bacterial or nuclease contamination. This can be prevented by storage at -20°C.
• DNA staining with methylene blue: Occasionally, ethidium bromide is not suitable for staining DNA. An alternative is methylene blue. In such cases, however, it must be kept in mind that the mass of DNA used must be increased by about 30 % and that about 1.5 h more must be planned for destaining steps. With a methylene blue concentrate supplied by AppliChem, you achieve very good results, even for small fragments of about 200 bp.
• Mass calculations for single fragments: Using DNA markers of a defined origin, the calculation of the mass of single fragments is relatively easy. The amount loaded per lane, e.g. 1 μg/10 μl, is divided by the number of base pairs of the DNA used and is multiplied by the fragment size. For example: the 267 bp fragment of the pBR322 Hae III marker with a loading amount of 1 μg: 1 μg : 4361 bp (pBR322) = 0.229 ng/base pair x 267 bp of the fragment = 61.2 ng/267 fragment.
• Less is often more: The different gel formats for agarose and polyacrylamide gel electrophoresis and the varying sensitivity of staining or detection mean that it is only possible to give an approximation of the recommended DNA amount to be loaded. Most DNA markers show the best separation with loading amounts of 0.5-1 μg on agarose gels. The general rule is: the lower the number of marker bands, the smaller the total amount needed. A low loading amount is also of advantage with short migration distances or very sensitive detection. When using polyacrylamide gels, generally only about 1/5-1/2 the amount required for agarose gels is necessary.
• How to achieve results quickly? Especially deproteinised DNA length markers can be separated very well using high voltages, e.g. within 20 minutes with a migration distance of 70 mm on a 1.2 % agarose gel at 120 Volt. This can only be realized, however, with a high quality gel migration buffer (the water quality must be taken into account!) and an adequate amount of buffer (400-600 ml). Poor buffer quality results in overheating of the agarose gel at higher voltages and this therefore causes problems. A high buffer volume (the buffer can usually be reused 4-6 times in one week without problems) leads the heat off, provided the gel chamber for submersion is of adequate size.
• Staining front too intense? In some cases, users who regularly perform assays with our products sometimes comment that the dye concentration in the gel loading buffer we supply is too high (only lyophilised markers). If this is the case, simply dissolve the DNA length marker in double concentration in the gel loading buffer and further dilute it with 1/2 volume TE buffer. Even when using a 50 % solution, there are no problems with the resulting 7 % glycerol concentration to increase the solution density.
• Non-plausible results (e.g. unexpected fragment runs at 500 bp instead of 350 bp) may occur under relatively extreme conditions (separation of restriction or PCR samples with a very high salt content > 150 mM) or at very high voltages with relatively short separation distances. To identify errors, 1/10 volume restriction buffer can be added to the aliquot of the DNA marker, for example, or the restriction sample can be appropriately diluted with gel loading buffer (usually 1/2 volume). In many cases, lowering the voltage during separation is also helpful. The accurate determination of the size of fragments or PCR products may also be impaired, when salt fronts combined with local problems of leading off heat with conducting away heat and very high electrophoresis voltages, result in partial denaturation of the DNA fragments. These are conditions that theoretically may also occur when processing samples from Maxam-Gilbert sequencing in combination with sample buffers containing formamide.