The Process of Ligation, Transformation, and Isolation of EGFP cDNA Into pET-41a(+) By Using Miniprep, Restriction Digest, PCR, and Bioinformatics To Ultimately Create Recombinant Expression Plasmids.
INTRODUCTION:
Scientists in this day and age are now able to change DNA and create recombinant DNA, which is a huge indication of great possibilities for the future of genetics. The goal of these lab experiments was to be able to create recombinant DNA with the insert of egfp gene. EGFP also known as Enhanced Green Fluorescent Protein was originally isolated from the jellyfish Aequorea Victoria and was introduced into the scientific field as a tool to signal gene expression. When EGFP is exposed to Ultraviolet Light, the protein glows a very …show more content…
The first two plates should have had green colony growth and possibly also white colony growth, plate #3 should have had green colonies, plate #4 should have no growth as results showed, plate #5 should have white colonies, and plate #6 should have bacterial lawn as results showed. With better and more accurate results, the transformation efficiencies would have changed as well with higher numbers in ug units.
Miniprep To Purify And Isolate plasmid DNA-

Figure 3: Miniprep Gel Electrophoresis. Figure 3 Key: Components of each well.
The photo shows the results of the Miniprep, which allows for the identification of the unknown sample. Well #3 (the unknown) has the highest band above the 10kb line, another band a little below the first band, and a band between 5-6kb. Well #4 (recombinant) shows similar bands as the unknown. The highest band is above the 10kb line, another band a little below the first band, and a band between 5-6kb. Well #5 has 3 bands above the 10kb line consisting of 1 band higher than Well #3 and #4’s highest band, a line very similar to the highest band in Well #3 and #4, and a band lower than the second band
Restriction Digest To Isolate egfp-
 …show more content…
Well #3 and #5 were very similar to each other with bandings at around 1.0-1.5kb, but also continual large lane banding. Well #4 of the non-recombinant has banding below 0.5kb. Well #6 was the Positive Control and showed one large lane band. Well #7 was the Negative Control that resulted in no bands because it was made up of H2O. The last well contained the DNA Ladder, which is used to measure the other band sizes.
Bioinformatics To Recreate An Online Recombinant Plasmid- The PCR product size was found to be 1183bp. The bold parts of the sequence at the start and end are the primers. A hypothetical Restriction Digest Design could use Acc65I and ArsI restriction enzymes to create cuts from the PCR product. The PCR product that was cut with only Acc65I would create 2 fragments of sizes 1-425bp and 426-1183bp. If the PCR product was cut with only ArsI, it would also create 2 fragments of sizes 1-723bp and 724-1183bp. The double digest would create 3 fragments of 1-425bp, 426-723bp, and
INTRODUCTION:
Scientists in this day and age are now able to change DNA and create recombinant DNA, which is a huge indication of great possibilities for the future of genetics. The goal of these lab experiments was to be able to create recombinant DNA with the insert of egfp gene. EGFP also known as Enhanced Green Fluorescent Protein was originally isolated from the jellyfish Aequorea Victoria and was introduced into the scientific field as a tool to signal gene expression. When EGFP is exposed to Ultraviolet Light, the protein glows a very …show more content…
The first two plates should have had green colony growth and possibly also white colony growth, plate #3 should have had green colonies, plate #4 should have no growth as results showed, plate #5 should have white colonies, and plate #6 should have bacterial lawn as results showed. With better and more accurate results, the transformation efficiencies would have changed as well with higher numbers in ug units.
Miniprep To Purify And Isolate plasmid DNA-

Figure 3: Miniprep Gel Electrophoresis. Figure 3 Key: Components of each well.
The photo shows the results of the Miniprep, which allows for the identification of the unknown sample. Well #3 (the unknown) has the highest band above the 10kb line, another band a little below the first band, and a band between 5-6kb. Well #4 (recombinant) shows similar bands as the unknown. The highest band is above the 10kb line, another band a little below the first band, and a band between 5-6kb. Well #5 has 3 bands above the 10kb line consisting of 1 band higher than Well #3 and #4’s highest band, a line very similar to the highest band in Well #3 and #4, and a band lower than the second band
Restriction Digest To Isolate egfp-
 …show more content…
Well #3 and #5 were very similar to each other with bandings at around 1.0-1.5kb, but also continual large lane banding. Well #4 of the non-recombinant has banding below 0.5kb. Well #6 was the Positive Control and showed one large lane band. Well #7 was the Negative Control that resulted in no bands because it was made up of H2O. The last well contained the DNA Ladder, which is used to measure the other band sizes.
Bioinformatics To Recreate An Online Recombinant Plasmid- The PCR product size was found to be 1183bp. The bold parts of the sequence at the start and end are the primers. A hypothetical Restriction Digest Design could use Acc65I and ArsI restriction enzymes to create cuts from the PCR product. The PCR product that was cut with only Acc65I would create 2 fragments of sizes 1-425bp and 426-1183bp. If the PCR product was cut with only ArsI, it would also create 2 fragments of sizes 1-723bp and 724-1183bp. The double digest would create 3 fragments of 1-425bp, 426-723bp, and