It is a method of range localization by physically interrupting the binding action of bacteria.
Operation method
basic program
Principle
In Escherichia coli cells, an exchange between the F factor and chromosomal DNA results in the insertion of the F factor into the chromosomal DNA of the host cell. Cells with an integrated F factor are called high frequency recombination (Hfr) cells. The location of the integration of the F factor varies in different Hfr strains. In Hfr bacteria and F-joining, the Hfr cell chromosome can enter the F-cell and undergo recombination.The transfer of the chromosome in Hfr bacteria starts from the end of the F-factor and can be interrupted at any time during the transfer process. Therefore, although F-bacteria received some Hfr genes after splicing, they generally could not receive F factors to become Hfr or F+. Due to the directionality and interruption of chromosome transfer, Hfr genes at the front end have more chances to appear in F-bacteria, and the genes at the back end have less chances to be transferred into F-cells. Therefore, according to the number of Hfr genes (i.e., the number of recombinants) in the F-bacteria after splicing, the order of gene transfer can be known. Thus, the relative position of genes on the chromosome, i.e., the order of genes on the chromosome, can be determined by the gradient transfer method.
Materials and Instruments
Donor: Escherichia coli CSH60: Hfr sup Acceptor: Escherichia coli E . coli) FD1004: F - leu purE trp his met A ilv arg thi ara lacY x yl m tl gal T6 r rif r strr Move 1. Inoculate the donor and recipient bacteria in 5ml LB liquid, and incubate overnight at 37℃ with shaking. 2. Transfer the overnight culture of donor and recipient bacteria into another diluted 5ml LB liquid in the ratio of 1:5, 37 ℃ continue to culture 2 ~ 3h. 3. 0.2ml of donor bacteria and 4ml of recipient bacteria were mixed in a 150ml sterile triangular flask, placed in a 37℃ water bath and gently shaken (50~100r/min). 4. respectively, in the culture of 0min, 10min, 20min, 30min, 40min, 50min when the above mixed bacterial solution sucked 0.2ml in a large test tube containing 10ml of sterile saline, immediately with a vibration mixer vibration vigorously for 30s, and then take 0.2ml in 3ml semi-solid medium with a vibration mixer mixing for 5s, poured in the choice of the plate on the level ( Each experimental group can choose one of the selective medium), while taking the donor bacteria and the recipient bacteria as a control (later sampling does not need to do the control), to be solidified and placed at 37 ℃ overnight incubation. For more product details, please visit Aladdin Scientific website.
LB culture solution A to D solid basic medium (one type for each group 6 dishes) Semi-solid agar Sterile saline
Constant temperature incubator Oscillating mixer Autoclave Petri dishes Triangular flasks Pipettes Test tubes