How to recover DNA from agarose gel?
Agarose or polyacrylamide gel electrophoresis is widely used as a high resolution technique for fractionation of DNA molecules by size. The basic methods for recovery of DNA from gel slices are (1) electroelution, (2) elution by diffusion, (3) gel dissolution, and (4) extrusion of DNA by gel compression.
What is low melting agarose what is its purpose?
Low melting temperature allows for the recovery of undamaged nucleic acids below denaturation temperature. Low Melting Agarose is ideal for digestion by agarose enzymes, which makes it very easy to recover large DNA fragments suitable for cloning or enzymatic processing.
How do you dissolve low melting agarose?
To melt the agarose in solutions of less than 2%, heat the slurry in microwave oven on high power setting until it starts to boil. Allow the solution to boil for 1 min or until the solution is clear and all particles are dissolved. Remove the flask from the microwave oven, and gently swirl to mix the agarose solution.
How does agarose gel concentration affect DNA migration?
The migration rates of DNA molecules in agarose gels are also affected by the composition of the gel. The migration rate of a DNA molecule decreases as the concentration of agarose in the gel increases.
How do you calculate DNA recovery?
The amount of DNA recovered = (the content of DNA after being recovered x the volume of the DNA after being recovered) / (the content of DNA before being recovered x the volume of the DNA before being recovered) x 100%.
How do you dissolve agarose gel?
It does take some time for the gel slice to completely dissolve. As it says in the protocol, you should heat the gel slice at 65*C and vortex briefly until it dissolves. You can add excess buffer if your gel is a high percentage. Make sure your agarose is completely dissolved or else it will clog up your column.
Can you remelt agarose gel?
Agarose can always be melted back down for reuse. Reusing low-melt agarose can influence concentrations since water is lost during the melting process. Aside from remelting, some researchers run the bands off the gel and reuse the gel without remelting.
What will not affect the rate of DNA migration through the agarose gel?
Question 3: Which of the following factors will not affect the rate of migration of DNA in agarose gels? CORRECT! Concentration of DNA will not affect the rate of its migration.
How to recover DNA from low melting point agarose?
Recovery of DNA from Low Melting Point Agarose Gels Run digestion products on 0.7% LMP agarose gel in 1X TBE (it’s nice to have at least 1ug of the fragment you want). LMP agarose is fragile; pour gel with EtBr Let solidify in cold room. Be sure to overlay the gel with buffer before pulling out the comb, to prevent damage to the wells
What’s the best way to re-melt agarose gel?
Re-melt the agarose by incubating at 65-70C for 5-10 minutes and immediately add 1 ml of buffer N2 prewarmed to >35C. Mix by vortexing and incubate at 37C until needed. (Alternatively, the gel slice can be placed in the N2 buffer first and then heated to 70C for 5-10 minutes or as required.
What’s the best way to remove agarose from DNA?
Cut out fragment of interest with clean razor blade and remove all excess agarose from the DNA. Use long wave UV to visualize fragment when curring as this reduces nicking. Don’t use UV box in the darkroom! Melt gel slice at 65oC 5min.
How are agarose gels used in molecular biology?
The use of low-melting-point (LMP) agarose gels has greatly facilitated many procedures in molecular biology. This type of agarose, although difficult to handle at concentrations less than 1.0%, has opened the door to fairly quick ways of isolating nucleic acids from gels without the need for electroelution, enzymes, or other methods.