The overall goal of this procedure is to visualize the DNA structural intermediates that occur when replication encounters DNA lesions using two dimensional gel analysis. This is accomplished by first isolating the DNA from actively dividing cells in which replication blocking lesions have been induced. Then gel electrophoresis is used to separate the recover DNA based on size.
Next, the lanes are cut out and recast horizontally in a second gel that further separates DNA based on both size and shape. Finally, southern analysis is used to visualize the DNA structures that are associated with replicating DNA fragments at times before and at various times after the introduction of UV induced damage. Ultimately, this procedure can be used to show that the inability to process DNA lesions in certain mutants results in accumulation of DNA repair intermediates.
Hi, my name is Arthur Ian. I'm from the Corella Lab at Portland State University. Hi, my name is Brand she, I'm also from the Corella Lab.
Today we're gonna show you a procedure for two dimensional gel electrophoresis. We use this procedure in our laboratory to study DNA replication and repair intermediates. So let's get started.
To begin this procedure, grow a 200 microliter overnight culture of e coli containing the plasmid PBR 3 22 in DGC thigh medium with ampicillin at 37 degrees Celsius. The use of DGC thigh medium minimizes UV shielding. Following overnight incubation pellet the cells at 14, 000 RRP M for 30 seconds.
Then resuspend the cell pellet in 200 microliters of DG C th medium lacking ampicillin and inoculate. 20 milliliters of DG C thigh medium grow cultures without ampicillin selection. In a shaking incubator at 37 degrees Celsius to an OD 600 of 0.5, which corresponds to about five times 10 to the eighth cells Per milliliter growth without ampicillin avoids selection against abnormal or unproductive replication intermediates that may arise in some mutants.
In addition, if using UV light to induce damage, the removal of the ampicillin from the media is necessary because it absorbs strongly at these wavelengths and shields. The cells reducing the effective dose of UV while the cells are growing. Use a UVC photometer to determine the distance from a 15 watt germicidal lamp that produces an exposure rate of approximately one JUUL per meter meter squared per second.
Subsequent steps should be performed under yellow light as this will prevent photo reactivation reversal of cyclo butane perine dimers by photo liase after UV irradiation. Once the desired cell density is reached, use a pipette to transfer the culture to a 15 centimeter diameter Petri dish on a rotating platform to ensure even irradiation of the entire cell population. Next, irradiate the cultures with 50 joules per meter squared and immediately transfer them into a sterile prewarm flask and place them back into the shaking 37 degrees Celsius incubator.
For the duration of the experiment, this dose produces on average one cyclo butane perine dimer, every 4.5 kilobases of single stranded DNA for each time point that is to be examined. Pipette a 0.75 milliliter aliquot of the culture into 0.75 milliliters of ice cold net 30. Buffer net 30 serves as a stop buffer that prevents replication and nucleotide excision repair from proceeding.
Once the stop buffer has been added, the sample can be kept on ice until the end of the time course. After the time course pellet each sample and resuspend the pellets. In 150 microliters of lysis buffer lyce the cells at 37 degrees Celsius for 20 minutes without agitation.
Because replicating structures with single strand regions or branch points are more susceptible to shearing cut pipette tips off with a razor blade to make the mouth wider and minimize shearing forces. In general, pipetting and agitation should be kept to a minimum until after the DNA has been digested with restriction enzymes, following lysis, add proteinase K and sarcas cell and allow the incubation to continue for one hour. This serves to release DNA fragments associated with protein.
Since actively replicating DNA is often bound to protein or membrane complexes, this helps increase the yield of replication fragments. After one hour, extract the samples by adding two volumes of phenol to each sample and gently inverting the tubes for five minutes. Then add two volumes of chloroform, isoamyl, alcohol, and gently invert the tubes for an additional five minutes.
Next, centrifuge the samples at 14, 000 RPM in a micro centrifuge for five minutes and transfer the top aqueous phase of each sample into a fresh tube. Then add four volumes of chloroform ISO alcohol, and again, gently invert the tubes for five minutes. Centrifuge the samples again at 14, 000 RPM for five minutes.
Place a dialysis membrane atop 250 milliliters of 0.1 xte in a beaker and spot 100 microliters of each sample onto the membrane. Let the samples dialyze for one hour after dialysis. Place 80 microliters of each sample into a fresh 1.5 milliliter tube containing 20 microliters of enzyme mix.
Digest the samples overnight at 37 degrees Celsius. PV two linearizes the plasmid just downstream from the origin of replication. Prior to loading the agros gel, add 100 microliters of chloroform and 20 microliters of six x loading dye containing bromo.
Phenol blue and xylene cyan to each sample and mix chloroform will strip any remaining PV two bound to DNA ends. Begin two dimensional aros gel analysis by running the restricted DNA samples through the first dimension. First load a Lambda hind three size marker into the first lane of a previously prepared 0.4%agros gel in one XTBE.
Then load 40 microliters of the aqueous phase containing the loading dye for each sample, skipping lanes to make gel slicing easier. Following electrophoresis, run the first dimension at one volt per centimeter for approximately 12 to 15 hours. The low voltage and low percent AGROS gel serves to separate the DNA fragments primarily based upon size.
After the first dimension has run, use a large butcher's knife to cut out the first lane containing the Lambda hind three marker and stain with atherium bromide. For the second dimension, slice out the gel lanes using the large butcher's knife using the Lambda Hind three marker as a guide crop and discard the region below where the linearized plasmid is expected to run on each lane. To cast the second dimension, place each slice horizontally across the top of an empty gel caster.
Prepare a solution of 1%AROS in one XTBE and cool to 55 degrees Celsius. Once cooled, pour the gel solution in to cast the second dimension, making sure to completely cover the gel slices. It is important to ensure the slices are evenly oriented and the castor's level before pouring the gel.
Once the gel has set, run the second dimension, five and a half to seven hours at 6.5 vols per centimeter in an electrophoresis unit that allows the buffer to recirculate the high voltage and high percent Agros gel effectively separates the DNA fragments based on their shape as well as their size. Non-linear shapes run more slowly through the second dimension following electrophoresis. Rinse the gel in water once and then wash it twice in 400 milliliters of 0.25 molar hydrochloric acid for 15 minutes with gentle agitation.
The acid washes serve to partially nick the DNA molecules into smaller fragments that transfer more efficiently during the southern analysis and are necessary due to the large size and unusual shape of the DNA replication intermediates. To prepare the gel for alkali transfer, rinse the gel again in water, then wash it twice in 400 milliliters of 0.4 molar sodium hydroxide for 30 minutes. The DNA in the gels is then transferred to a high bond n plus nylon membrane using a downward alkali transfer system with 0.4 molar sodium hydroxide as described in the written portion of this procedure, let the DNA transfer for six to 12 hours following DNA transfer continue with probe hybridization as outlined in the written protocol.
Once the membrane has been probed and washed, place the membrane on a paper towel until the liquid has visibly disappeared. Then wrap the membrane in polyvinyl chloride plastic wrap and expose it to a phospho imager screen. Finally, the radioactivity can be visualized and quantified using a storm eight 40 and its associated image quant.
The migration pattern of PV two digested PBR 3 22 plasmid observed by 2D aros gel analysis is diagrammed. Non replicating linear plasmids run as linear 4.4 KB fragment replicating plasmid form Y shaped structures that migrate slower than the non replicating fragments due to their larger size and non-linear shape. This migration pattern forms an arc that extends out from the linear region towards the well following UV irradiation.
Double Y or X shaped molecules are observed in the cone region and migrate more slowly than the arc of Y shaped molecules. An example of the southern analysis of the 2D gel probed with P 32 labeled PBR 3 22 is shown for cells immediately after UV irradiation and 15 minutes after UV irradiation immediately after UV irradiation. Approximately 1%of the total plasma DNA can be found in the Y arc when cells are rapidly growing in exponential phase following a radiation.
A transient increase in YS shaped molecules is observed as blocked replication forks accumulate at damaged sites. The L-shaped replication intermediates also transiently, accumulate and persist until a time that correlates with when the lesions are repaired. We've just shown you how to visualize DNA repair intermediates using two dimensional gel electrophoresis.
When doing this procedure, it's important to be aware of shearing forces that can reduce yield and to be gentle with all samples and gels. So that's it. Thanks for watching and good luck with your experiments.
