Nucleic acid Electrophoresis
The electrophoretic behavior in gels of DNA and RNA is effected by their size and shape. Both RNA and DNA can exist as single and double stranded molecules and the single stranded forms have significant secondary and tertiary structure. For this reason single stranded nucleic acids are best separated as denatured structures. This is most often an issue for ssDNA after sequencing reactions, and RNA which can be denatured by urea, methylmercuric hydroxide, formamide, formaldehyde and a number of other agents.
Agarose is a highly purified naturally occurring polysaccharide and preparation of agarose gels involves simply heating the powdered agarose in buffer to dissolve it. It gels upon cooling. Like acrylamide, the pore size of an agarose gel is inversely dependent on the agarose concentration. The pores in agarose gels are generally much larger than those in acrylamide gels and are widely used in separation of nucleic acids. Low molecular weight nucleic acids and oligonucleotides, however, are usually separated by PAGE, due to smaller pore size of the gel matrix.
Agarose gels are simply formed by placing the necessary weight of agarose in the required volume of buffer. This is then heated to boiling to dissolve the agarose. A microwave oven is the fastest way to do this. Before casting the gel the agarose solution is cooled down to 50 °C.
There are many different types of agarose available. The best choice for routine DNA electrophoresis is Agarose SERVA Wide Range (cat.no. 11406) or Agarose for DNA electrophoresis (cat. no. 11404). This offers good gel strength and low impurities that might interfere with subsequent procedures. Other qualities like Agarose SERVA for PCR (cat. no. 11383) are made for efficient separation of small PCR generated DNA fragments. It has a high gel strength for better handling and enhanced visibility due to improved clarity of the gel. For easy recovering of DNA fragments after electrophoretic separation a range of low melting agaroses are available.
For size determination of DNA fragments in agarose gels you need size markers of high quality under the respect of fragment size and purity.
SERVA offers two types of DNA MW size markers:
SERVA DNA standard lyophilized
- Range covers traditional MW standards made by digestion of pUC19, pBR328 or phage λ DNA as well as 100 bp and 1 Kbp ladders for PCR fragment analysis
- High-quality fragment ends, lyophilized - can be resuspended in buffer of choice for labelling experiments, e.g. fill-in, 5’-end label
- Stable for at least 3 years (if stored at -20 °C), 1 x 1 ml sample buffer is included for easy and fast resuspension of the DNA fragments
- Range covers ready-to-use, supplied in loading buffer DNA ladders for fragment ranges from 50 – 1500 bp, 100 – 3000 bp and 250 bp – 25 Kbp
- For estimation of DNA mass of bands of similar size with comparable intensity, the approximate mass of each band is indicated
- Stable for 6 months at 25 °C and for 12 months at 4 °C. For long term storage store at -20 °C
SERVA DNA Stain G is a safer alternative to traditional ethidium bromide stain for detecting nucleic acid in agarose gels. It is at least as sensitive as ethidium bromide and can b...
SERVALYT™ Carrier Ampholytes SERVALYT™ Carrier Ampholytes are low molecular weight molecules of zwitterionic character. They are a mixture of synthetically derived species of avera...
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DOWEX® Ion Exchange Resins DOWEX® resins are made of spherical particles. The benefit for the user is a highly efficient exchange process, due to optimal flow-through kinetics bec...