Presentation on theme: "Standard Protocol for PCR Product Clean-up and Electrophoresis 洪禎憶."— Presentation transcript:
Standard Protocol for PCR Product Clean-up and Electrophoresis 洪禎憶
PART Ⅰ : Standard Protocol for PCR Product Clean-up
Introduction The Gel/PCR DNA Fragments Extraction Kit is designed to recover or concentrate DAN fragments (50bp-10kb) from agarose gel 、 PCR or other enzymatic reaction. The method uses a chaotropic salt, guanidine thiocyanante to dissolve the agarose gel and denature enzymes. The DNA fragments in the chaotropic salt are bound to the glass fiber matrix of the spin column. After washing off the contaminants, the purified DNA fragments are eluted by a low salt elution buffer or water. Salts, enzymes and unincorporated nucleotides can be effectively removed from reaction mixture.
Gel extraction protocol 1.Gel dissolving: About 300mg of the gel slice add 500ml of DF buffer. Incubate at 55 ℃ ~60 ℃ for 10-15min until the gel has been completely dissolved. During incubation, invert the tube every 2- 3min. 2.DNA binding: Place a DF column in a 2 ml collection tube. Apply the sample mixture into the DF Column. Centrifuge at 8000rpm for 1 min. Discard the flow. 3.Wash: Add 600λ of wash buffer (ethanol added) in the DF column.Centrifuge at 8000rpm for 1 min. Discard the flow- through and place the DF column back in the collection tube. Centrifuge again for 3 min at 13,000 rpm to dry the membrane of column. 4.DNA elution: Transfer dried DF column on a clean microcentrifuge tube. Add 20λ of elution buffer directly onto the center of the membrane in the DF column. Stand for 2 min until elution buffer is absorbed. Centrifuge for 3 min to elute purified DNA.
PCR clean up protocol 1.Sample preparation: Add 5 volumes of DF buffer and mix. 2.DNA binding: Place a DF column in a 2 ml collection tube. Apply the sample mixture from previous step into the DF Column. Centrifuge at 8000rpm for 1 min. Discard the flow-through and place the DF column back in the collection tube. 3.Wash: Add 600λ of wash buffer (ethanol added) in the DF column.Centrifuge at 8000rpm for 1 min. Discard the flow- through and place the DF column back in the collection tube. Centrifuge again for 3 min at 13,000 rpm to dry the membrane of column. Transfer the dried DF column on a clean microcentrifuge tube. 4.DNA elution: Transfer dried DF column on a clean microcentrifuge tube. Add 20λ of elution buffer directly onto the center of the membrane in the DF column. Stand for 2 min until elution buffer is absorbed. Centrifuge for 3 min to elute purified DNA.
5 X DF Buffer
96 Well Gel/PCR DNA Extraction Kit 1.DNA binding: Transfer PCR products to of clean V-bottom plate plate. Add 250λ Binding buffer to each well and mix by pipetting. Place a DNA binding plate on top of the vacuum manifold. Transfer the sample mixture to each well of DNA binding plate. Apply vacuum at 10 inches Hg for 5 min until wells have emptied. 2.Wash: Add 250λ of Wash buffer (ethanol added) to each well of DNA Binding Plate. Apply vacuum at 10 inches Hg for 5 min until wells have emptied. Add 250λ of Wash buffer (ethanol added) to each well of DNA Binding Plate to wash again. Apply vacuum at 10 inches Hg for 5 min until wells have emptied. Press the DNA Binding plate on an absorbent material (tissue paper) to blot out the excess liquid from the bottom of the plate. 3.DNA elution: Centrifuge at 3500rpm for 10 min to remove ehanol residue. Transfer the DNA Binding Plate on a new storage plate. Add 50λ of elution buffer in the center of the membrane. Stand for 2 min until elution buffer absorbed by the matrix. Centrifuge for 5 min at 3500 rpm in a centrifuge to elute purified DNA.
96 well plate
Part Ⅱ : Introduction of Gel Electrophoresis
Introduction Agarose gel electrophoresis is a method for separating, identify, and purify DNA fragments. The fragments are separated by charge and size, by forcing them to move through a agarose gel matrix which is subjected to an electric field. The electric field is generated by applying voltage across a buffer.
Protocol To pour a gel- 2% - 3.2g agarose powder add 160ml TAE buffer for PCR products. 0.8% g agarose powder add 160 ml TAE buffer for plasmid DNA. After the gel has solidified, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells. DNA will migrate towards the positive electrode, which is usually colored red. DNA fragments are visualized by staining with ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator.
Agarose Gel Agarose is a linear polymer composed of alternating residues of D-galactose joined by α(1 3) andβ (1 4) glycosidic linkage. Chains of agarose form helical fibers that aggregate into supercoiled structures.
Migration of DNA Fragments in Agarose The molecular size of the DNA -Largr molecules migrate more slowly. The concentration of agarose -Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. 1000bp
The conformation of the DNA -Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. The presence of EtBr in the gel - Intercalation of ethidium bromide causes a decrease in the negative charge of DNA and increase its mass and rigidity, and therefore its mobility.
Electrophoresis Buffer -DNA fragments will migrate at somewhat different rates in TAE or TBE buffer. In the absence of ions, there will be essentially no migration of DNA in the gel. In buffer of high ionic strength, enough heat may be generated in the gel to melt it. The applied voltage -To obtain maximum resolution of DNA fragments ＞ 2kb in size, agarose should be run at no more than 5-8V/cm. The type of agarose -The two major classes of agarose are standard agaroses and low-melting-temperature agaroses.
Electrophoresis buffers Several different buffers are available for electrophoresis. These contain Tris-acetate and EDTA(TAE), Tris-borate(TBE), Tris-phosphate(TPE). TAE and TBE -TAE has the lowest buffering capacity and will become exhausted if electrophoresis is carried out for prolonged period of time. Better resolution of supercoiled DNAs. linear, double stranded DNA runs faster. -TBE has better buffer capacity. -TBE and TPE both are slightly more expensive.
Gel loading buffers Gel loading buffer are mixed with samples before loading into the slot of gel. These buffers serve three purposes: 1.They increase the density of the sample, Ensuring that the DNA sinks evenly into the well. 2.They add color to the samples, thereby simplifying the loading process. 3.They contain dyes that, in an electric field, move toward the anode at predictable rates.
Ethidium Bromide Ethidium Bromide (EtBr), 3,8 diamino-5-ethyl-6-phenyl- phenanthridium bromide, is used as a marker for identifying and visualizing nucleic acids. Bands contain as little as ~10ng of DNA can be detected in the presence of free ethidium bromide in the gel. Ethidium bromide can be used to detect both single- and double-stranded nucleic acids (both DNA and RNA).