This method can help answer key questions in molecular and cellular biology field, such as identifying homologous recombination events in mouse embryonic stem cells. The main advantage of this technique is that it is accurate, reliable, and widely used. So, this method can provide insight into identifying homologous recombination events in mouse embryonic stem cell.
It can also be applied to other systems, such as genetically modified cell or animals. Generally, individual new to this method will struggle because it take long time and involves multiple steps. For each gDNA, mix 30 microliters of 10 x buffer for DRA one, three microliters of DRA one and 10 micrograms of gDNAs per sample.
Add water until the final volume is 30 microliters, and incubate at 37 degrees celsius overnight to digest the gDNAs. Prepare a 1%agarose electrophoresis gel with ethidium bromide. Load the previously acquired samples along with a one KB ladder onto the gel, and run it at 30 to 40 volts overnight.
The next day, soak the gel in a tray containing a 0.2 newton hydrochloric acid solution. Use a shaker to shake the tray gently for 20 minutes at room temperature. Transfer the gel to a tray containing a DNA denaturing solution.
Shake it gently for 20 minutes at room temperature. Then transfer the gel into a tray containing a DNA neutralizing solution. And shake it gently for 20 minutes at room temperature.
Using a rapid downward transfer system, transfer the DNAs from the gel to the membrane. Next, assemble the turble blotter and blotting stack as outlined in the instructions provided by the manufacturer. After this, take out the membrane and wash it with two X saline sodium citrate for one minute.
Absorb the liquid with tissues, and then use a UV crosslinker to cross link the DNA with the membrane. To begin labeling the DNA probes with radioactivity, add the heat denatured probe DNAs to the tube containing the ready to go DNA labeling beads. Pipette up and down to mix.
And add five microliters of radioactively labeled deoxycytodine triphosphate. Incubate at 37 degrees celsius for 15 minutes. Purify the labeled probes by using G 50 micro columns according to the instructions provided by the manufacturer.
Use a scintillation counter to measure the radioactivity. To begin hybridizing the membranes with the labeled probes, place the membrane into the hybridization tube. Add the mixed pre hybridization solution.
Place the tube into hybridization oven and let the pre hybridization proceed at 42 degrees celsius for 30 minutes. Then, remove the tube from the oven and pour the pre hybridization solution into a 50 milliliter tube. Add the denatured probe, and mix gently.
To remove the non hybridized probes, place the membrane into a tray containing one X saline sodium citrate with 0.1%SDS and shake gently at 55 to 60 degrees celsius for 10 minutes. After this, wrap the membrane with plastic wrap and fix it in the exposure cassette. In a dark room, expose the membrane to two sheets of X ray film.
Develop the film to visualize the results. Determine if a corresponding ES clone is the desired one with the target of recombination or not, according to the sizes of the DNA bands detected by the probes. In this study, southern blotting and PCR is utilized to identify HR events that occur in mouse ES cells for the generation of NM two genetic replacement mouse models using ES cells based HR mediated targeting technology.
Because the genomic DNAs are cut into many fragments with different lengths, they display a smear like status on the DNA gel. This suggests a complete digestion of the genomic DNAs. As a final step of southern blotting, the signals of a radioactivity labeled probe hybridizing with a target DNA fragment are shown on the film.
The appearance of expected bands reflects the occurrence of HR events in the ES clones. According to the predesign in the study, ES clones with mutated allele have two distinct size bands. While wild type ES clones only have one band, suggesting the desired ES clones are heterozygous.
Following the PCR reaction, the PCR products can be analyzed on the DNA gel. If a specific NPCR band of the correct size is observed, this suggests the occurrence of HR events which can be further confirmed by sequencing that PCR band. The implication of this technique extended to the therapy of identifying point mutation because the foundation is identical.
While attempting this procedure is important to remember to be patient and careful. Following this procedure, other method like microinjection of ES cell into blastocysts can be performed in order to answer additional question like there formation of chimeric animals. Aft its development, this technique paved the way for the researcher in their field of molecular and cellular biology to explore the function of a specific gene or the consequence of a mutated gene in mice or beyond.
Don't forget that working with radioactivity labeler P32 DCTB can be extremely hazardous and precautions such as shielding with protection board should be always be taking while performing this procedure.
