By Stefan Surzycki
This laboratory guide offers a radical advent to trendy, expense effective recommendations in Molecular Biology, which may locate software in lots of diverse fields. it's the results of useful adventure, with each one protocol having been used commonly in undergraduate classes and workshops or proven within the author's laboratory. hence, also they are prone to paintings in green fingers the 1st time they're performed.
step by step protocols and useful notes are supplied. The distinguishing characteristic of this guide in comparison to others is the particular rationalization of the theoretical mechanisms of every step and the dialogue of the significance of the recommendations. moreover, each one protocol contains a sign of the time and price interested in its program. this data will permit the clients to layout their very own variations or to evolve the tactic to varied structures.
Dr. Surzycki has been instructing undergraduate classes and major workshops in introductory Molecular Biology for a few years, within which time he has largely changed and subtle the strategies he has defined during this manual.
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Additional resources for Basic Techniques in Molecular Biology
This DNA can be used for all molecular biology procedures. 1 General Aspects of DNA Isolation and Purification Promega Co. corn) Promega offers several Wizard" Genomic DNA Purification Kits each designed for isolation ofDNA from different sources (blood cells, culture cells and animal tissue, plant tissue, yeast, and gram positive and gram negative bacteria). These procedures use a four-step process: cell lysis, RNase digestion, and removal of proteins by salt precipitation and concentration of DNA by isopropanol precipitation.
The first procedure is a "hybrid" between the phenol and chloroform extraction methods, with the addition of Proteinase K digestion. The theoretical rationale for each procedure is described in detail in Chapter 1. Cells are washed in PBS and resuspended in cell lysis buffer containing Sarcosyl, EDTA and NaCl. 12 M, respectively. The majority of proteins are removed by Proteinase K treatment. The Proteinase K concentration used is very high (1000 ug/rnl) to shorten the time of the treatment, and the volume is small (about 10 ml), to decrease the cost of treatment.
Samples with an absorbance equal to or larger than 2 should be diluted before measuring. 2) can be strongly affected by light scattering on dust particles present in the preparation. Measuring the absorbance at 320 nm (Schleif and Wensink 1981) should assess the degree of such contamination. At this wavelength, DNA does not absorb and any absorbance at 320 nm is due to light scattering. The absorbance at 320 nm should be less than 5% of the A260• Absorbance measurements at wavelengths other than 260 nm are used for determination of DNA purity.