By Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)
Read Online or Download A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA PDF
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Extra info for A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA
Notes: STEP-By-STEP PROCEDURE 65 6 Immobilization of DNA on Nylon Membranes The Immobilizing Matrix Two common types of membrane suitable for nucleic-acid hybridization are currently on the market: nylon and nitrocellulose filters. The nylon membranes, because of their greater physical strength are generally preferred to the nitrocellulose membranes. Genomic sequencing requires nylon membranes of highly homogeneous composition. However for technical reasons it seems that the manufacturers of nylon membranes are not yet able to produce membranes of the quality required for genomic sequencing.
A weight placed on the top of the anode ensures a tight contact between the gel and the membrane (Saluz and Jost, 1986). INTRODUCTION 59 during the transfer time is guaranteed without perturbing the velocity of the DNA molecules to be transferred, and the volume of the buffer should be large enough so that its temperature is constant over the transfer time. The best results were obtained with the system shown in Fig. , 1981) is easily constructed, cheap and very efficient. It consists of a lower sieve plate electrode (cathode), a layer of Whatman 17 paper, the sequencing gel, a nylon membrane, a second layer of Whatman 17 paper and an upper sieve plate electrode (anode).
0S g bisacrylamide (recrystallized twice) > 16S g urea (analytical grade) > 40 ml of 10 x TBE buffer > Distilled water to 400 ml > Dissolve the ingredients into solution by mixing with a magnetic stirring bar. 2 ml of 10 % freshly prepared ammonium persulfate and mix well. > Filter the solution through Schleicher & Schuell, No. Ls 14112 filter paper. > Degas the solution in a Buechner flask under vacuum for at least 5 minutes (shake bottle gently from time to time). tI ofTEMED. > Mix gently. > Pour the gel as indicated in Fig.
A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)