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Five Tips & Products for Avoiding Mishaps in Your Molecular Biology Applications
Tip 1: Agarose
Agarose gel electrophoresis remains the most widely used technique for separating nucleic acid fragments due to non-toxicity and a broad separation range. Following a few simple steps during gel casting can help you avoid burns and achieve best resolution.
- Always use a flask 2–4 times the volume of the solution. Allow the powder to hydrate in the solution for a few minutes before heating. Weigh the flask and solution before and after heating and add warm distilled water to solution to bring it to its original weight.
- To avoid bubble formation, cool the hot agarose solution to 50–60oC and pour carefully into gel cassette. Allow gel solution to cool gradually; rapid cooling will cause an irregular gel matrix and band distortion during electrophoresis.
- When using low melting agarose gel, it is important to run the gel in a cold buffer. High voltage can cause overheating of the buffer, which can melt the gel. The UltraPure™ Agarose products are made from the highest purity biochemicals and packaged in a pouch that makes pouring and dispensing the powder easier—reducing the likelihood of spills and contamination.
Tip 2: Electrophoresis Buffer
The most commonly used buffers for DNA electrophoresis are TAE (Tris-acetate EDTA) or TBE (Tris-borate-EDTA). DNA fragments will migrate at different rates in these two buffers due to differences in ionic strength.
Typically, the TAE buffer provides better resolution of fragments greater than 4 kb, and the TBE buffer resolves 0.1–3 kb fragments. The TBE buffer is better suited for highvoltage (>150V) electrophoresis because of its higher buffering capacity and lower conductivity. If you mistakenly use water instead of buffer, there will essentially be no migration of DNA in the gel. Avoid mishaps with conveniently packaged, ready-to-use 10x TBE or 10x TAE Buffer.
Tip 3: Ultrapure Water
Laboratory water has multiple uses in the research labs, from glassware rinsing to highly sensitive applications involving biological samples. Unfortunately, in many labs, water contains many contaminants, such as trace organics, particles, bacteria, and, importantly, nucleases, all of which can have a detrimental effect on the resulting date.
For example, if nucleases are present in PCR reaction, they will effectively cleave any DNA, including the primers and probes. Amplification will therefore not occur. How pure is the source of water used in your PCR reaction? Why not eliminate possible contaminates in your PCR reaction by trying UltraPureTM DNase/RNase Free Distilled Water? With no DNase, RNase, or protease activity detected, it’s designed for use in all molecular biology applications.
Tip 4: DNA Ladders
An essential tool used during gel electrophoresis is a DNA marker or ladder, which is used to determine the size of an unknown DNA sample. DNA ladders are easy to identify because they contain regularly spaced, distinct-sized samples that, when run on an agarose gel, look like a ladder.
The recommended loading volume is dependent on detection methods: Typically, one ng DNA band can be detected with ethidium bromide staining. However, we normally load different volumes—e.g., 10 ul and 2 ul of a DNA ladder in the first and last lanes of a gel to orient the gel and to increase the quantity range. Use of two tracking dyes in the DNA ladders and samples ensures the DNA bands will not run off the gel and indicate maximum resolution has been achieved. Life Technologies offers a wide range of DNA ladders in the ready-to-load TrackItTM format. The TrackItTM DNA Ladders are ready to use—no need to heat, mix, or dilute.
Tip 5: DNA Stain
The most common dye used to make DNA or RNA bands visible is ethidium bromide (EtBr).
It fluoresces under UV light when the dye intercalates with DNA or RNA. Short exposure of nucleic acids to UV light can cause significant damage to the sample, which will reduce the efficiency of subsequent manipulations of samples, such as ligation or cloning.
If DNA is to be used after separation on agarose gel, it is best to avoid exposure to UV light by using a blue light excitation source and stain, such as SYBR Safe DNA Stain. SYBR Safe DNA Stain is a highly sensitive stain for visualization of DNA that has been shown to have a low level of mutagenicity and toxicity. It is classified as non-hazardous waste with similar sensitivity as EtBr.
For a limited time, we are offering UltraPure™ product essentials, DNA ladders and DNA stains at a special discounted price.
For UltraPure reagents, use promotion code MH0202 when placing your order*.
For DNA ladders, use promotion code P862959 when placing your order*.
For DNA stains, use promotion code P862950 when placing your order*.
Hurry! Offers expire September 14, 2012.
UltraPure™ reagents for electrophereisis | UltraPure™ reagents for protein isolation | UltraPure™ reagents for cloning | UltraPure™ reagents for hybridization | UltraPure™ reagents for general chemical | UltraPure™ reagents for nucleic acid purification | DNA ladders | DNA stains
Terms and Conditions:
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
*This promotion is open to academic end users in Hong Kong from July 13 to September 14, 2012. Discount will apply to orders received by Life Technologies Limited no later than September 14, 2012 or until promotional supplies are depleted, whichever comes first. Discount applies to list price in effect at the time order is received by Life Technologies. Cannot be combined with other discounts or promotions. Offer void where prohibited, licensed or restricted by federal, state, provincial, or local laws or regulation or agency/institutional policy. Other restrictions may apply. Life Technologies Limited reserves the rights at its sole discretion to determine the eligibility to receive the special offer.