TH12409C3 - The process of building a fosmid library of living organisms. - Google Patents

Abstract

DC60 (24/06/16) This invention aims to improve the process of building fosmid libraries in living organisms. To be another option that has no complicated steps This will reduce the working time by 1-2 days, thus reducing the use of labor, chemicals and costs. While the quality of the Fosmid library is still good at an acceptable standard for future use of the Fosmid library. The invention based process can be applied to the DNA of All living things, whether they are micro-genome organisms such as microbes, can be used in large-genome organisms such as plants and animals. The process consists of the following steps: DNA extraction. A of the target organism Doing DNA of the target organism To be smaller by physical shearing to get DNA Suitable size for cloning Preliminary DNA size was assessed on agarose gel, DNA selection of target organisms on agarose gel. To get the required size DNA By separating the DNA on the gel Agarose and DNA was cut on agarose gel in the range of 30-60 kB. DNA extraction of target organisms size as required. Want to leave the gel agarose With the electroelution method (Electroelution) DNA making of target organisms size DNA concentration by means of DNA precipitation with ethanol and sodium acetate, DNA end repair of target organisms of desired size (DNA end repair) ) To obtain a suitable DNA tip to connect to a DNA carrier (vector) with a blunt end. DNA piece welding of living things Target of the desired size, fixed end, with a phosphid dNA carrier (ligation) to create a phage library, phage packaging, library migration. Phosphid enters Escherichia coli bacteria. The phosphid library is stored in Bacterial storage plate at -80 ° C. Edit Summary of Invention 24/06/2016 This invention aims to improve the formation process. Fosmid library in living things In order to be an alternative way with no complicated steps This will reduce the working time by 1-2 days, thus reducing the use of labor, chemicals and costs. The quality of the Fosmid library is still good at an acceptable standard for future use of the Fosmid library. This invention can be applied to the DNA of All living things, whether living organisms with small genomes such as microorganisms, can be used in large genome organisms such as plants and animals. The process consists of the following steps: DNA extraction. Of the target organism DNA making of the target organism To be smaller by means of images (physical shearing) to get DNA Suitable size for cloning Preliminary DNA size was assessed, agarose gel, DNA selection of target organisms on agarose gel. To get the required size DNA By separating the DNA on the gel Agarose and DNA was cut on agarose gel in the range of 30-60 kB. DNA extraction of target organisms size as required. Want to leave the gel agarose With the electroelution method Electroelution) DNA making of target organisms size DNA concentration by means of DNA precipitation with ethanol and sodium acetate, DNA end repair of target organisms of desired size (DNA end repair) ) To obtain a suitable DNA tip to connect to a DNA carrier (vector) with a blunt end. DNA piece welding of living things Target of the desired size, fixed end-of-the-way with a phage packaging, a phage packaging, a migration. Phosphid libraries enters Escherichia coli bacteria Storing phosphid libraries in Bacterial storage plates at -80 degrees Celsius ------------------- ----------------------------------------------- This invention It is intended to improve the process of building fosmid libraries in living organisms. In order to be an alternative way with no complicated steps This will reduce the working time by 1-2 days, thus reducing the use of labor, chemicals and costs. The quality of the Fosmid library is still good at an acceptable standard for future use of the Fosmid library. The invention based process can be applied to the DNA of All living things, whether organisms with small genomes such as microorganisms, can be used in large genome organisms such as plants and animals. The process consists of the following steps: DNA extraction. Of the target organism Minimizing the DNA of the target organism by physical shearing to obtain DNA Suitable size for cloning Preliminary DNA size was assessed, agarose gel, DNA selection of target organisms on agarose gel. To get the required DNA size by separating the DNA on the gel. Agarose and DNA cut on agarose gel in the 30-60 kB range. Extraction of target organism DNA required size from agarose gel. With the electroelution method Electroelution) Intensification of the target organism's DNA concentration (DNA concentration) with the method of DNA precipitation with ethanol and sodium acetate. Target organisms of the required size (DNA end repair) to obtain a suitable DNA tip to connect with DNA vectors with blunt end. Linking DNA pieces of target organisms of the required size that were fixed at the end of the repair to the phosphid-type DNA carrier (ligation) to build the phosphid library. Phage packaging Phage packaging Migration of phosphid libraries into Escherichia coli Bacterial storage plates at -80 ° C.

Claims (2)

Disclaimer (all) which will not appear on the advertisement page: edit 24/6/2016 1. The process of creating a phosphid library of which consists of the following steps: 1.1 Extraction of the target organism's DNA, can be selected from the preparation method in the form of a DNA embedded in a gel (plug) or a Prepared with cetyl trimethyl ammonium bromide; CTAB or a preparation with Any one of the alkaline lysis (alkaline lysis). 1.2 DNA of the target organism. To be smaller by physical shearing, selectable from pipetting or adding cooling and reducing cooling (freeze thaw cycle) in order to obtain a suitable DNA in the Cloning Preliminary DNA size was assessed on agarose gel. 1.3 DNA selection of target organisms on agarose gel. To get the required DNA size This was done by separating DNA gel agarose and cutting DNA on agarose gel in the range of 30-60 kilobases. 1.4 DNA extraction of target organisms of desired size from agar gel. Rose By electroelution 1.5, the DNA concentration of the target organisms of the desired size is increased (DNA concentration) by means of precipitation of the DNA with ethanol and sodium A. Cetate 1.6 DNA end repair of target organisms of the required size (DNA end repair) to obtain a suitable DNA tip in connection with the end of the DNA vector. The blunt side was 1.7 to weld the DNA piece of the target organism of the required size that was repaired at the end to We took a phosphid ligation to build a fosmid library 1.8 Filling a phosphid library into a phage packaging 1.9 Transfer of a phosphid libraries into Escherichia coli bacteria. 1.10 Storage of phosphid libraries in Bacterial storahe plate at -80 ° C. 2. The process of building a phosphid library of organisms according to claim 1, where the target organisms can be selected from plant or animal or microorganisms. The one -------------------------------------------------- ---------------------- 1. The process of creating a phosphid library of living organisms, which consists of the following steps: 1.1 Extraction of Organism DNA. Target life 1.2 Doing the DNA of the target organism To be smaller by physical shearing to obtain suitable DNA cloning. The preliminary DNA size was assessed for agarose gel. 1.3 DNA selection of target organisms on agarose gel. To get the required size DNA This was done by separating the DNA on agarose gel and cutting the DNA on agarose gel in the range of 30-60 kB. 1.4 DNA extraction of the target organisms of the desired size from the gel. Rose Using electroelution method) 1.5 Intensification of the target organism's DNA concentration (DNA concentration) by precipitation of DNA with ethanol and sodium acetate. 1.6 DNA end repair of target organisms of the required size (DNA end repair) to obtain a suitable DNA end in connection with DNA vector with a blunt end. Yes, 1.7 welding of DNA pieces of target organisms of the required size to be repaired at the ends and then to Fosmid carrier DNA (Ligation) to build the fosmid libraries 1.8 Filling the phosphid libraries in the phage packaging 1.9 Transfer the phosphid libraries into Escherichia coli bacteria 1.10 Storing the phosphid library in a Bacterial storage plate at -80 degrees Celsius.