At this point, the cells on the filter membrane are lysed in order to open up the plasmids for easier access and their DNA is denatured, which allows it to bind to the filter. The bacterial colonies are then symmetrically replicated onto the nitrocellulose filter by direct contact. 45 μm, undergoes these processes to ensure that there is no contamination during the transfer, thus allowing for accuracy in results. A nitrocellulose filter is then washed three times with distilled water, placed in between absorbent sheets, and heated at high temperatures to kill bacteria or other microorganism. These bacterial plasmids are cultured on a nutrient agar plate, leading to the formation of bacterial colonies, some of which ideally continue to contain the gene of interest.
A specific piece of DNA is removed from its respective cell culture and inserted into a bacterial plasmid via a process known as recombination. Methods Ĭolony hybridization begins with a desire to extract a segment of DNA containing a specific gene, such as a gene that conveys antibiotic resistance. This method was discovered by Michael Grunstein and David S. The most common purpose of colony hybridization is to verify that a certain DNA sequence was able to successfully enter into a new cell, meaning that the cells being analyzed through this method are the result of recombination between a specific piece of DNA and a bacterial plasmid. Radiographed RNA is used to find the desired sequence within the new bacterial colony and essentially "light it up" so that the sequence can be identified for transfer. The overall process involves a transfer of genetic material from one medium to another, typically using nitrocellulose filter paper, with the intended goal of identifying and isolating a specific gene. The genes of interest have been added to a bacterial plasmid previously through recombination, allowing genes from other organisms to be analyzed within a bacterial colony. Wrap the membrane in plastic wrap and expose the membrane to X-ray film in a film cassette at -80☌ for autoradiography (or place the wrapped membrane on a phosphoimager screen also see Hint #5).The process of colony hybridization: growth of cell colonies, replication on filter, hybridization, and identification of desired colonies.Ĭolony hybridization is a method of selecting bacterial colonies with desired genes through a straightforward cloning and transfer process. After washes are complete, remove the membrane from the container with a pair of forceps. Discard the solutions after each incubation and monitor the radioactivity present on the membrane between incubations.ġ0.
Remove Wash Buffer 2 from the last incubation in Step #8 and incubate two times with 50 ml of Wash Buffer 3 for 10 min at 37°. Discard the solutions after each incubation and monitor the radioactivity present on the membrane between incubations.ĩ. Remove Wash Buffer 1 from the last incubation in Step #7 and incubate the membrane three times with 50 ml of Wash Buffer 2 for 20 min at 65☌. Discard the solutions after each incubation and monitor the radioactivity present on the membrane between incubations.Ĩ. Once the hybridization is complete, carefully remove the Hybridization Solution and incubate three times with 50 ml of Wash Buffer 1 for 10 min at 65☌. Allow the hybridization to continue for 6 hr or more to overnight (see Hint #4).ħ. Add the DNA probe to the hybridization bottle to obtain a final concentration of approximately 100,000 to 500,000 cpm/ml of Hybridization Solution (see Hint #3).Ħ. Snap cool the DNA probe by immediately placing the tube containing the probe in an ice bath for 5 min.ĥ. Boil the radiolabeled DNA probe for 3 min to denature the DNA (see Protocol #872 CAUTION! See Hint #2).Ĥ. Pre-hybridize the membrane at 60☌ (for Northern blots) or 65☌ (for plaque hybridization or RNA dot blots) for at least 1 hr with gentle agitation in a rotary hybridization oven.ģ. Place the membrane to be probed in a hybridization bottle with a minimum amount of Standard Hybridization Solution (0.1 ml per square cm of membrane also see Hint #1).Ģ.
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