DNA analysis by
By Condy Kan
Introduction
Osamu Shimomura and his colleagues successfully discovered and isolated green fluorescent protein (GFP) from the jellyfish Aequorea victoria; GFP consists of a tripeptide, which is Ser-65, Tyr-66, and Gly-67, that forms fluorescence chromophore (1). Fluorescence chromophore, is accountable for its green fluorescence under UV light, helps to identify and determine the cells if the Enhanced Green Fluorescent Protein (EGFP) gene is expressed or not (2). In addition, Isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer for bacteria E. coli since it binds to lac repressor of the EGFP gene and the lac operator activates T7 promoter that promotes the transcription of EGFP gene (3). EGFP is …show more content…
In the ligation reactions, recombinant DNA plasmids contained egfp fragments that were inserted with pET-41a(+) vectors with the restriction endonucleases and DNA ligase. Next, the ligation products were transformed into cultured E. coli which were taken as three samples: recombinant plasmids, non-recombinant plasmids, and unknown plasmids; the transformation of E. coli went through ice incubation and heat shock. Further, the plasmids were isolated and purified by using the "Wizard" mini-columns and then digested with restriction digest reactions: double digest and single digest. The products were compared with the sample without any enzymes; finally, Polymerase Chain Reaction (PCR) was used to amplify purified DNA. Overall, in the ligations, miniprep, restriction digest reactions, and PCR experiments, every sample was analyzed and identified by using gel electrophoresis. In one experiment, transformations of E.coli were plated on the wide variety of petri dishes and left to be incubated overnight in order to observe green fluorescent bacteria colonies over using the UV …show more content…
Ligation #1 was consisted of 1:1 molar ratio of pET-41a(+) vector to egfp insert with 50 ng NcoI/NotI cut pET-41a(+) DNA, 7 ng egfp insert DNA, sterile dH2O, and 10x ligase buffer, and DNA ligase. Ligation #2 contained a 1:3 molar ratio of pET-41a(+) vector to egfp insert with 50 ng NcoI/NotI cut pET-41a(+) DNA, 21 ng egfp insert DNA, sterile dH2O, and 10x ligase buffer, and DNA ligase. In the case of positive control, Ligation #3 was contained 50 ng uncut pET-41a(+)/EGFP recombinant plasmid DNA and sterile dH2O. Both Ligation #4 and Ligation #5 were used as a negative control; Ligation #4 only contained sterile dH2O and Ligation #5 contained 50ng NcoI/NotI cut pET-41a(+) DNA, 21 ng egfp insert DNA, sterile dH2O, and 10x
By Condy Kan
Introduction
Osamu Shimomura and his colleagues successfully discovered and isolated green fluorescent protein (GFP) from the jellyfish Aequorea victoria; GFP consists of a tripeptide, which is Ser-65, Tyr-66, and Gly-67, that forms fluorescence chromophore (1). Fluorescence chromophore, is accountable for its green fluorescence under UV light, helps to identify and determine the cells if the Enhanced Green Fluorescent Protein (EGFP) gene is expressed or not (2). In addition, Isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer for bacteria E. coli since it binds to lac repressor of the EGFP gene and the lac operator activates T7 promoter that promotes the transcription of EGFP gene (3). EGFP is …show more content…
In the ligation reactions, recombinant DNA plasmids contained egfp fragments that were inserted with pET-41a(+) vectors with the restriction endonucleases and DNA ligase. Next, the ligation products were transformed into cultured E. coli which were taken as three samples: recombinant plasmids, non-recombinant plasmids, and unknown plasmids; the transformation of E. coli went through ice incubation and heat shock. Further, the plasmids were isolated and purified by using the "Wizard" mini-columns and then digested with restriction digest reactions: double digest and single digest. The products were compared with the sample without any enzymes; finally, Polymerase Chain Reaction (PCR) was used to amplify purified DNA. Overall, in the ligations, miniprep, restriction digest reactions, and PCR experiments, every sample was analyzed and identified by using gel electrophoresis. In one experiment, transformations of E.coli were plated on the wide variety of petri dishes and left to be incubated overnight in order to observe green fluorescent bacteria colonies over using the UV …show more content…
Ligation #1 was consisted of 1:1 molar ratio of pET-41a(+) vector to egfp insert with 50 ng NcoI/NotI cut pET-41a(+) DNA, 7 ng egfp insert DNA, sterile dH2O, and 10x ligase buffer, and DNA ligase. Ligation #2 contained a 1:3 molar ratio of pET-41a(+) vector to egfp insert with 50 ng NcoI/NotI cut pET-41a(+) DNA, 21 ng egfp insert DNA, sterile dH2O, and 10x ligase buffer, and DNA ligase. In the case of positive control, Ligation #3 was contained 50 ng uncut pET-41a(+)/EGFP recombinant plasmid DNA and sterile dH2O. Both Ligation #4 and Ligation #5 were used as a negative control; Ligation #4 only contained sterile dH2O and Ligation #5 contained 50ng NcoI/NotI cut pET-41a(+) DNA, 21 ng egfp insert DNA, sterile dH2O, and 10x