The germs are interbred in place.

method may be used when cloning
a small number of
(100-200) scattered on several
agar tablets. These places are combined into a agar main plate and a nitric cellulose membrane that has been placed on the surface of the second agar plate. After
culture
after a period of time, the bactericides were cracked in place. The main plate should be stored at 4 degrees C until the screening results are obtained..1. Transfer a small number of bacteriosphase to the cellulose nitrate membrane:
(1) Place a nitric cellulose membrane on an agar plate containing selective
antibiotics
.
(2) The individual flora is transferred to the membrane with a sterile toothpick and then to the agar main plate containing selective antibiotics but not the filter film. Line vaccinations (or dots) should be performed in a certain grid. Each microsecosis should be crossed in the same position as the two plates. Finally, a bacteriostrim containing non-recombined prosurfic particles (e.g. pBR322) is zoned on both the membrane and the main plate.
(3) inverted plate, cultured at 37 degrees C to the line of bacterial germs grow to a width of 0.5-1.0mm.
(4) the filter film is pierced with a syringe needle filled with waterproof black drawing ink until agar is marked in more than 3 asymmetric positions. Mark at roughly the same position on the main tablet.
(5) seal the main plate with a Palafilm membrane and store it upside down at 4 degrees C until the result of the hybrid reaction is obtained.
(6) cleavage bacteria, according to the method described below this paragraph, so that the release of
. DNA
in the cellulose nitrate membrane..2. The cleavage of the bacterium and DNA combined with the nitric cellulose membrane
(1) on a cling film to make a small depression (0.75 ml) containing 0.5mol/L NaOH, so that the bacterioslation face up, put the membrane on the small depression, flatten the cling film, so that the membrane is evenly moist, leaving the membrane in place for 2-3 minutes.
(2) Use a dry paper towel to absorb the membrane from the bottom of the membrane and repeat the procedure (1) with a new cling film and a newly made 0.5mol/L NaOH.
(3) drain the membrane and transfer it to a new one with 1mol/L Tris. Cl (pH7.4) on the cling film depression. After 5 minutes, absorb the membrane and repeat the procedure.
(4) suction filter film and transfer it to 1.5mol/L NaCl, 0.5mol/L Tris. Cl (pH7.4) of the cl cl cling film after 5 minutes of absorbent membrane, transferred to a dry
filtration paper
, placed at room temperature for 20-30 minutes, so that the membrane
dry
.
(5) clamp the membrane between two dry sheets of filter paper and bake dry in a vacuum oven for 2 hours at 80 degrees C to secure the DNA.
(6) interbreeds dna fixed to the membrane with a 32 P-labeled probe..3.
(1) with 2×SSC plastic plate, the dry roasted membrane floating on the liquid surface, thoroughly soaked for 5 minutes.
(2) transfer the membrane to a glass dish with 200 ml of pre-washed liquid. How the membranes are stacked together and put in the solution. Cover the glass dish with cling film and place on a rotating platform located in the culture box. Process for 30 minutes at 50 degrees C. In this and all later steps, the membranes should be shaken slowly to prevent them from sticking together.
(3) Gently wipe bacterial fragments from the membrane surface with water-absorbing cotton paper soaked with pre-washed liquid to reduce the hybrid background without affecting the strength and clarity of the positive hybrid signal.
(4) Transfer the membrane to a glass containing 150 ml of prebreeding fluid, prebreeding for 1-2 hours at the appropriate temperature (i.e. 68 degrees C when interbreeding in an aqueous solution and 42 degrees C when interbreeding in 50% methamide).
(5) Heat the 32 P-labeled double-stranded DNA probe at 100 degrees C
for
5 minutes and quickly place it in an ice bath. Single-stranded probes do not have to be denatured. Add the probe to the hybrid bag and crossbreed overnight. During hybridization, containers containing membranes should be tightly covered to prevent liquid from evaporating.
(6) after hybridization, remove the hybrid fluid and immediately place the membrane at room temperature in a large volume (300-500 ml) of 2×SSC and 0.1%
. In the
SDS, gently shake for 5 minutes and flip the membrane at least once. Wash again and again, while avoiding the film from drying up.
(7) 68 degrees C with 300-500ml 1×SSC and 0.1% SDS solution wash membrane twice, each time 1-1.5 hours. Radiation self-development is now available. If the background is very high or the experimental requirements are strict film washing conditions, the membrane can be soaked at 68 degrees C for 60 minutes using a solution of 300-500 ml 0.2×SSC and 0.1% SDS.
(8) After drying the filter film on a paper towel at room temperature, place the filter film (numbered face up) on a cling film, and make several asymmetric markings on the cling film so that the membrane corresponds to the radioactive self-display film position.
(9) cover the membrane with a second cling film. Add X-rays and an add-on screen for 12-16 hours of exposure at -70 degrees C.
(10) negative is shown, attach a clear piece of hard paper to the negative. Mark the position of the positive hybrid signal on paper and at the same time mark the position of the asymmetric distribution point. Transparent paper can be removed from the negatives to identify positive bacteria by comparing the points on the paper with between the points on the agar fat. .