Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size.
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer. The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide.
The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.
The most important factor is the length of the DNA molecule, smaller molecules travel farther.
Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid. The most common dye used for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr.
Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well.
Gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules are only able to move end-on in a process called "reptation" and are more difficult to separate. After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA bands.
Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ.
A color marker dye containing a low molecular weight dye such as "bromophenol blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA solution more dense so it will sink into the wells of the gel). After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also highly recommended). Intro: UV TransilluminatorUV-transilluminators are used in molecular biology labs to view DNA (or RNA) that has been separated by electrophoresis through an agarose gel. Step 6: Assemble the hinged safety lidIn the final assembly steps we will add a hinged safety lid.
For the UV transilluminator enclosure and lid, download the design file attached below (svg or PDF file). Instead of creating BioBricks, as is the IGEM project norm, we focussed on creating novel ways for existing BioBricks to be used by ordinary citizen scientists to investigate their neighborhoods.
We also borrowed some techniques of grassroot cartographers to build our own maps of the soil samples and map GEL bands on to them. We collected various soil samples from which we extracted DNA and ran it through a PCR machine (using standard Biobrick primers) followed by gel electrophoresis to characterize the combined flora and fauna of a site visually; the ecology’s fingerprint, if you will. 3.After the protein is removed the DNA can be precipitated using cold ethanol or isopropanol (propan-2-ol). 6.Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light. 1.For dissolving the DNA present in the soil, mix the soil sample (5 g) in Tris Buffer (5 mL of 25 mM buffer) and mix thoroughly. 2.Then in order to separate the cells out of the solution, filter using 5?m cellulose nitrate filter. 4.The pellet, containing the concentrated DNA (after centrifugation) is separated from the supernatant, and dissolved in 100?L autoclaved milliQ water.
1.For dissolving the DNA present in the soil, mix the soil sample in Tris Buffer (2 gm soil sample in 2 mL 25mM Tris Buffer) and mix thoroughly. 2.Freeze the eppendorf tubes containing the above mixture in liquid nitrogen, and then boil (at 100°C for 20 mins). 5.Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light. 1.Add the tris buffer to the agarose powder, heat at 50 degrees centigrade till you obtain a clear solution and add Ethidium bromide.
2.Pour into the Gel box, add the comb to form wells, get rid of any air bubbles and let it settle for 30 minutes. Measure out the weights of tryptone, yeast extract and sodium chloride as above then fill up with deionised water to 1L and mix well until clear. Measure out the weights of tryptone, yeast extract, sodium chloride and agar with deionised water to 1L and mix well.
Add the sybrsafe™ (10?l) pour the gel into the mold and leave it to set for 15 minutes.
Inject your DNA samples into the appropriate wells and use a HyperLadder for reference (left hand side). Turn on the machine and make sure the black lead is attached to the black end and the red lead is attached to the red end.
Autoclave both solutions separately to avoid the reaction of glucose with other components. Keep solutions at 4 oC overnight, and LB at 37 oC so that when cells get transferred they do not experience a temperature change. Put cells on ice for 10 mins (keep cold from now on, and cool everything on ice before adding).
If using DNA from the kit plates, resuspend DNA in the well in 10 ?l water, pipetting up and down. With X-tracta, DNA sample extractions can be performed with great accuracy and precision in a rapid, four-step process.
The X-tracta agarose gel extraction tool provides a fast, convenient, precise and safe way to extract and dispense samples.
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Strains with our parts, the positive and negative controls were cultured in a 24-well microplate in M63 Mannitol during 24H at 30°C. These results show that the percentage of adhesion is similar between the strains containing the three parts and the positive control, thus tagged CsgA were still functional.
The supernatant was removed and the remaining biofilm was fixed to the microplate by heat treatment at 80°C during 1H. Crystal violet staining shows that the strain containing the three parts could form a biofilm like the positive control. Strains with our parts, the positive and negative control were cultured in M63 Mannitol at 30°C and 180rpm. Congo Red staining shows that the CsgA with one or two tags expressed by the P70 promoter allows to form curli fibers which are able to bind Congo Red. For the Confocal Laser Scanning Microscopy biofilm acquisitions, all the strains were cultivated in 96-well microplate in M63 Mannitol during 16H at 30°C (See Protocol for details).
As no strain carrying our parts showed a significant difference with the positive control, our part’s insertion doesn’t modify the biofilm formation properties.
For the Transmission Electron Microscopy, all the strains were cultured in conditions that allow curli formation: 48h (for a 50 mL culture), 28?C temperature and low agitation.
The bacteria cultures we used are a csgA-knockout strain as negative control, and the three engineered csgA constructions, the WT, the His1-Tag and His2-Tag.
The images show that there is no significant difference between our positive control and our constructions. The expression of CsgA derivatives expressed by the p70 csg promoter carried by the psb1c3 plasmid leads to functional CsgA, and allows E. Then, liquid cultured strains were assayed for biofilm nickel absorption using the calibration curve, after measuring the OD of the complex formed for each strain at 554nm. Although quantification is possible, this technique lacks precision and is more suited for qualitative studies. Nickel-DMG complex colorimetry measurement follows a linear regression for concentrations from 20uM to 100uM.
A second method has been used, more quantitative and more precise (but more expensive) : ICP-MS (Inductively coupled plasma mass spectrometry). To address biosafety issues linked with GMOs, we worked on destroying our bacteria after letting them grow in a biofilm. Wells containing M63 cultures of strain 227 were put under UV light or exposed to heat treatments (at 60 or 70°C) for different lengths of time.
Epifluorescence observations were made after Back Light coloration of 227 strain liquid cultures. As a follow-up to the exploration of curli production and nickel chelation, we want to know the kinetics behind the 70 base-pair long promoter sequence that we used during the whole summer. On a pKK backbone, two essential parts have been assembled: a promoter and a reporter gene. On the one hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP, the construction is called p70:GFP.
On the other hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation region of curli production and combined to the reporter GFP, the construction is called P750:GFP.
During the early growth stages at 37°C, we can observe that the P70 (orange) has a higher expression level compared to the P750 (red). We conclude that P70 has the ability to prematurely activate downstream expression at 37°C. However at 30°C, P70 (light blue) stabilizes within the range of low expression levels and P750 (dark blue) reaches the highest promoter expression rates.
We conclude that P750 has a delayed, albeit extremely high-leveled, promoter expression at 30°C. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). In general higher concentrations of agarose are better for larger molecules; it will exaggerate the distances between bands.
Different purities of agarose are commercially available as are agaroses with different melting properties. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white (see figures). A solution of up to 4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.8% is preferable. Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The DNA moves toward the positive anode due to the negative charges on its phosphate backbone. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. Based on work by Scott Williams, Jacki Buros (bot) and Alexandra Almonacid, wikidoc user MDas and wikidoc anonymous user Statsone.
During or immediately after electrophoresis, the agarose gel is stained with a fluorescent dye which binds to nucleic acid.
Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.
Do not leave the UV light on for too long before taking the photo as this can degrade the DNA. The disposable polyethylene tool cleanly cuts and picks up uniform slices of gel, eliminating possible cross-contamination from razor blades and other gel-cutting methods. To also remove yourself from searches for specific user names, you will need to set your Flickr profile to be hidden from searches.
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The supernatant was removed and the OD600 measured, then the bacteria forming the biofilm were resuspended and the OD600 was measured in order to estimate the number of cells (See protocol for details).

CsgA with one or two tags expressed by the P70 promoter were sufficient to form thick biofilms. The crystal violet solution was added in each well in order to stain the cells and the wells were washed with water to remove crystal violet in excess (See protocol for details). After centrifugation, the supernatant was removed and the cell pellet was resuspended in the Congo Red solution, in order to specifically stain the curli. Dimethylglyoxime (DMG) was used as a complexing reagent, which forms a pink-colored complex (peak absorption at 554nm) in the presence of Ni(II).
That means that only two His-tags on C-term can improve the natural nickel chelation capacities of CsgA . As the captured metal is extracellular and Curli proteins are very resistant to environmental changes, alive bacteria are not needed for our biofilter. Well contents were then gradually transferred into Eppendorfs and diluted (100, 300, 900 and 2700 fold). From the solution obtained, Curli promoter(750 bp) was amplified by PCR with Q5 polymerase and designed primers.
However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream. To avoid this problem linear molecules are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.
Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a constant amount while length and breadth are varied as needed). Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running. Make sure the gel is completely covered with TBE, and that the slots are at the end electrode that will have the negative current. The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel. If you do not have access to a laser cutter, you can send the files to any laser cutting service such as Pololu.
And if I have some luck in finding this type of a lamp: the question is how to disperse the light of this lamp, to illuminate sample properly?
The photographs from these were sown together to produce a comprehensive map of the immediate surroundings. We did this by vortexing using phenol-chloroform, and further the addition of SDS (a detergent) to remove the membranes of lipids.
The corresponding positive and negative controls are, respectively, Wild-type E.coli curli producing strain transformed with the empty vector and csgA--knockout E. The samples were centrifuged again and the pellets were observed (See protocol for more details). Moreover, our results show that the addition of one or two His-Tag on the C-term of CsgA doesn’t disturb the normal properties of curli (sturdiness, adhesion, structure and folding of CsgA). The quantity of chelated nickel for each strain was compared to the quantity of curlis formed by each strain.
They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel.
Instead these gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.
The vast majority of agarose gels used in modern biochemistry and molecular biology are prepared and run horizontally. This technique is used wherever the researcher needs to be able to view their sample, for example sizing a PCR product, purifying DNA segment after a restriction enzyme digest, quantifying DNA or verifying RNA integrity after extraction.
Materials for laser cutting can be found at any supplier of acrylic materials (McMaster-Carr, US Plastics etc) except for the solacryl (UV-transmissive) which can be bought from Loop Acrylics.
It can be explained by the conformation of the His1-tag which could be folded on the side of CsgA, as presented in the modelization section. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp. It is now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light.
This Instructable tutorial describes how to make a UVB (310nm) transilluminator with a 7 x 7 cm window for viewing ethidium bromide (or SYBR-Safe) stained DNA mini-gels. Potentially, further increase of the amount of His-tags could improve the nickel accumulation capacities of CsgA. Great job in particular explaining how to use the professional-grade 312nm UVB bulb and ballast (only $28 for the bulb! Other users are strongly recommended to use SYBR-Safe instead, which can be handled and disposed of more safely. I have found that 2424 Blue Acrylic works as prefilter and 2422 Orange Acrylic works as a postfilter for those stains. However, when the lid is not in place, safety glasses mustbe worn when operating the UVB bulb.
The glass is cut to your custom size, at least it was for me anyway, and cost $40 per 70x70mm part. However, they had a minimum order of at least 3 parts, so really it was $120 for the 3 pieces.

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