Deletion localization is a gene localization method in which a strain at the mutation position to be tested is recombined separately with a series of mutant strains with known deletion regions, and the position of the deletion in the mutant strain to be tested is determined based on whether or not it can be recombined.
Operation method
basic program
Principle
If a deletion mutation strain can be recombined with a known deletion mutation strain, the position of the mutation must not be within the known deletion region. If recombination is not possible, the position of the mutation in the strain to be tested is within the known deletion region. The deletion regions of strains A, B and C are known, and there are a series of point mutation strains 1, 2, 3 and 4, which are added dropwise to the same position on the surface of the solid medium, according to whether the wild-type recombinants appear or not (indicated by the + sign), the position of the point mutation can be known. If the missing fragments of strains A, B and C are in the range indicated by the dotted lines in the figure below, then the order of the four point mutations is 1432 or 1342. The chromosomes indicated by the solid lines at the ends of the dotted lines here are actually connected. When it is added with strain 3, wild-type recombinants appeared, but when it is added with strain 4, no wild-type recombinants appeared, so it can be further confirmed that the order of the mutation site is 1432 rather than 1342, which shows that as long as there are enough deletion strains, it is possible to differentiate the mutation sites that are very close to each other. Deletion localization can be applied not only to general gene localization studies, but also to the localization of mutation sites of a gene, i.e., fine structure analysis of genes. The efficiency of deletion localization can be improved not only by selective culture method, but also by observing only the results of recombination or not, without observing the number of recombinants. Therefore, this gene localization method is not only efficient but also accurate. Of course, it is necessary to obtain a set of deletion mutant strains before applying this method. In this experiment, the fine structure of lacZ gene was localized. We used a set of F-strain with a known nonsense mutation in the lacZ gene of E. coli chromosome and a set of strains with a lacpro chromosome fragment on F′ and different lengths of deletions in the lacZ gene. Since the F- strains used in the experiments were trp-strr and the donor bacteria with F′ were strs, tryptophan and streptomycin could be added to the lactose-containing basal medium in order to select the recipient bacteria and exclude the donor bacteria. The dropwise hybridization technique can be easily observed on this medium to analyze the position of the mutation or the length of the deletion in the lacZ gene of these two sets of strains, i.e., the localization of the point mutation or deletion mutation.
Materials and Instruments
Escherichia coli Move 1. Donor and recipient bacteria were connected to 5 ml of LB culture medium and incubated at 30℃ for overnight shaking. 2. Dropwise hybridization test was carried out on relatively dry basic medium containing lactose, tryptophan and streptomycin placed overnight at 30℃ or 37℃, 12 dishes in each group. Each plate was divided into 9 compartments according to Figure 34-3, each labeled with 12, 13, 14, ..., 19, and the 9th compartment was labeled with ① or ②, ③, ④, ..., 1 and a blank control. 12 to 19 were the donor organisms, CSH12, CSH13, and ......, respectively, CSH19, and the number in the circle is the recipient strain number. Recipient ① was added dropwise to 9 areas of plate ①, and recipient ② was added dropwise to 9 areas of plate ② until plate 1, and no recipient was added to the blank control plate. Use the inoculation ring in each plate marked with 12 in the area of the donor bacteria 12 drops, each plate marking the area of the donor strain 12 drops, then add the donor strain 13, until the donor strain 19, the blank control plate does not add drops of the recipient strain, only the donor strain 12 to 19. until the surface of the plate drops of the liquid dry, placed in 37 ℃ incubation for 24 ～ 48h. 3. Observation results Caveat 1. The plate for dropwise hybridization should be properly dried, so as to facilitate the rapid drying of the bacterial solution. 2. In the drop with the recipient bacteria on the plate and then add the donor bacteria, after each addition of the inoculation ring must be flame-sterilized, so as not to mix a bacterial solution into another hybridization area. For more product details, please visit Aladdin Scientific website.
LB Liquid Medium Lactose Tryptophan Streptomycin Solid Medium 12 Dishes
Autoclave Constant Temperature Incubator Petri dish Straws