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For better DNA gel extraction results, minimize exposure to UV light as much as possible
UV light is notorious for damaging DNA. Cut your gel slice quickly. When working with multiple bands, trim one at a time on the UV as opposed to letting the UV light damage the bands that are waiting to be cut. Alternatively, use a more visible range stain instead of UV light.
The best way to determine the purity of my DNA?
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure the purity of the sample. This is because DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength can be used to estimate the DNA concentration using an equation derived from “Beer’s Law”. A ratio of ~1.8 is generally accepted as pure for DNA, whereas a ratio of ~2.0 is generally accepted as pure for RNA, however as the aromatic amino acids, tyrosine and tryptophan absorb strongly at 280 nm, a decrease in the ratio is used as an indicator of protein contamination.
The A260/A230 is another indicator of contaminants, the expected values are commonly in the range of 2.0-2.2, Contaminants such as EDTA, carbohydrates, phenols and have an absorbance close to 230 nm and so will decrease the ratio, whereas guanidine isothiocyanate will increase the ratio. A poorer ratio is therefore often found when extracting using nucleic acid purification columns, although this may not interfere with downstream processes.
Agarose gel electrophoresis is a common molecular biology technique used to separate DNA or RNA molecules by size. This is achieved by denaturing and applying a negative charge to our nucleic acid sample. The samples are then run through an agarose matrix with an electric field (Electrophoresis). Shorter molecules move faster and thus migrate further and in the gel over a given time period. Many factors are involved with regards to the final visualization step and achieving high resolution of the resulting bands on the agarose gel.
Tips to help improve resolution include: - Running the gel at a lower voltage for a longer period of time - Using a wider/thinner gel comb - Loading less DNA into each well
MangoTaq comes with a coloured reaction buffer that contains red and orange dyes, which separate during electrophoresis and provide quick reference points for monitoring the mobility of the DNA samples in the gel.
Real-Time PCR has become an increasingly popular technique for analysis of gene expression. There are two primary methods of real-time PCR that can be performed. The first involves including the reverse transcriptase step in the same tube as the PCR reaction (one-step).
The second method involves creating cDNA first by means of a separate reverse transcription reaction and then adding the cDNA to the PCR reaction (two-step). There are advantages and disadvantages to both systems that you should considered before choosing the best one for your application, these include the ease of use and cost of reaction to the resulting yield and sequence representation.
An RNase free environment is essential when working with RNA samples. Obtaining full-length, high-quality RNA can be a real challenge in the lab.
The structure and lability of RNA, and the fact that enzymes that degrade RNA are ubiquitous, hardy, and nearly impossible to remove can make working with RNA difficult. Even trace amounts of RNase can degrade RNA, so it is essential to avoid inadvertently introducing RNase into RNA samples during or after the isolation procedure.
To help you ensure the best possible results when working with RNA, we've published some key hints and tips for working with RNA samples.
The article explores the main reasons for RNA degradation and discusses the various sources of RNases such as skin, dust, reagents and the samples themselves. It also includes suggestions on lab precautions you can take such as wearing gloves, using RNase inhibitors, and explains how high-quality reagents will help maintain an RNase-free environment.