Analysis of DNA Using Restriction Enzyme Digestion
Objective: DNA is analyzed by agarose gel electrophoresis after being digested with EcoRI restriction endonucleasse.
Procedures:
λ DNA and puC18 DNA were put into two tubes respectively. Then, EcoRI buffer, EcoRI enzyme and deionized water would be put into both tubes. EcoRI enzyme was the restriction enzyme that cut the DNA at the specific sequence. The EcoRI buffer enhanced the stability of many enzymes and binds contaminants that may be present in DNA preparations. DI water was used to bring the solution into a required volume for gel electrophoresis. The prepared sample was incubated at 37℃ for overnight. 40mL of a 0.8% agarose gel is prepared after the preparation of the samples was done. Agarose and 1X TAE electrophoresis buffer were added into an Erlenmeyer flask. The 1X TAE electrophoresis buffer provided the correct pH for the gel to run. The solution would be heat until the agarose is completely dissolved. Ethidium bromide solution would be added to the solution after it cooled slightly. Ethidium bromide solution was a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments. It enabled visualization of the fragments within the gel under UV light. The solution would be poured into the gel mold for solidification. The gel was stored at 4℃ until used.
All the samples and the gel were allowed to come back to room temperature. The gel was transferred into the electrophoresis chamber and covered with 1X TAE buffer. The 1X TAE electrophoresis buffer provided the correct pH and it gave ions to support the conductivity for the gel to run. The uncut λ DNA would be added to labeled tube as a control DNA. By comparing the conformations of uncut DNA with the cut DNA, the completed digestion could be verified. Molecular marker, which was used for molecular weight calculation, was stored in another sample tube. The prepared sample contained puC18 DNA would be run as the degrade DNA control
Procedures:
λ DNA and puC18 DNA were put into two tubes respectively. Then, EcoRI buffer, EcoRI enzyme and deionized water would be put into both tubes. EcoRI enzyme was the restriction enzyme that cut the DNA at the specific sequence. The EcoRI buffer enhanced the stability of many enzymes and binds contaminants that may be present in DNA preparations. DI water was used to bring the solution into a required volume for gel electrophoresis. The prepared sample was incubated at 37℃ for overnight. 40mL of a 0.8% agarose gel is prepared after the preparation of the samples was done. Agarose and 1X TAE electrophoresis buffer were added into an Erlenmeyer flask. The 1X TAE electrophoresis buffer provided the correct pH for the gel to run. The solution would be heat until the agarose is completely dissolved. Ethidium bromide solution would be added to the solution after it cooled slightly. Ethidium bromide solution was a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments. It enabled visualization of the fragments within the gel under UV light. The solution would be poured into the gel mold for solidification. The gel was stored at 4℃ until used.
All the samples and the gel were allowed to come back to room temperature. The gel was transferred into the electrophoresis chamber and covered with 1X TAE buffer. The 1X TAE electrophoresis buffer provided the correct pH and it gave ions to support the conductivity for the gel to run. The uncut λ DNA would be added to labeled tube as a control DNA. By comparing the conformations of uncut DNA with the cut DNA, the completed digestion could be verified. Molecular marker, which was used for molecular weight calculation, was stored in another sample tube. The prepared sample contained puC18 DNA would be run as the degrade DNA control