Colony PCR and DNA Mini-Prep
Colony PCR:
Two white colonies and one blue colony was chosen from the plate that contained the negative control for the ligations. Then five major colonies and one blue colony was chosen from the plate that contained the positive control DNA ligation. 5 white colonies were chosen from the plate that contained E. coli that had vectors that contained the 533/1492 inserts. Four major white colonies were chosen from the plate that contained E. coli cells that should have contained vectors with the 515/806 inserts. Then, five white colonies were selected from the plate containing E. coli cells with the vectors that had the NS1/NS2 inserts. Each of the colonies chosen were inoculated into a tube of 100µL of nuclease free water. Then 24 PCR tubes were taken and labeled 1-24. 7.5µL of nuclease free water was added to each tube, and then 12.5µL of the Taq master mix was carefully added to each tube. Following the Taq master mix, 2.5µL of the M13 F/R was added to each tube. Then 3µL of the inoculated E. coli cells were appropriately added to specific tubes. Colonies grown on the plate containing the negative control were added to tubes 1-3. The colonies that were present on the positive control plate were added to tubes 4-9 and the colonies that contained the 533F/1492R inserts were added to tubes 10-14. For tubes 15-18, the colonies that contained 515F/806R were added and for tubes 19-24, the colonies containing the NS1/NS2 inserts were added. Then each tube was run through a Thermocylcer. For the initial denaturing, the temperature was increased to 95ᵒC for 5 minutes. It remains at 95ᵒC for another 45 seconds and then the temperature is decreased to 50ᵒC for 1 minute for the primers to anneal. After 1 minute, the temperature is raised to 72ᵒC for the extension of the DNA for one minute and then the temperature is raised to 95ᵒC once again for 45 seconds. The cycle repeats 34 times. After 34 cycles, the temperature is raised to 72ᵒC once more for 10 minutes for the final extension. The samples are then stored at 4ᵒC. To measure the concentration of the DNA, Each sample was run through a Thermo Scientific Nanodrop 2000 Spectrophotometer.
DNA Mini-Prep:
Eight microcentrifuge tubes were labeled as MC1-MC8. 1 ml of culture was added to the tubes which were kept on ice at 4 degrees Celsius. The remainder was centrifuged at 14,000x g for ten minutes. The bacteria was re-suspended by adding 250 µl of the R3 solution and mixing it until the solution became homogeneous. 250 µl of the solutions labeled as L7 was added to the solution and it was mixed by inverting the solution using a micropipette. Then it was incubated at room temperature for five minutes. 350 µL of the solution labeled as N4 was then added to the microcentrifuge tube. The tube was then centrifuged at 14,000x g for ten minutes. After ten minutes, all of the supernatant was loaded onto a spin column which was placed in a wash tube. The column was centrifuged at 12,000x g for one minute. Then the flow through that was collected in the wash tube was discarded. 500 µL of W10 was then added to the column, and the column was incubated for one minute at room temperature. The column was then centrifuged at 12,000x g again for one minute. After centrifuging, the flow through in the wash tube was discarded. Then 700µL of the solution labeled as W9 was added to the column. The column was then centrifuged at 12,000x g for one minute twice and each time, the flow through was discarded. Then, the spin column was placed in a recovery tube and the wash tube was thrown away. 75µL of the solution labeled as TE was added directly to the center of the column and incubate for one minute at room temperature. Then centrifuge at 12,000x g for two minutes. Throw away the column. The flow through in the recovery tube should contain pure DNA. To find the concentration of the DNA, the DNA was run through a Thermo Scientific Nanodrop 2000 Spectrophotometer.
Two white colonies and one blue colony was chosen from the plate that contained the negative control for the ligations. Then five major colonies and one blue colony was chosen from the plate that contained the positive control DNA ligation. 5 white colonies were chosen from the plate that contained E. coli that had vectors that contained the 533/1492 inserts. Four major white colonies were chosen from the plate that contained E. coli cells that should have contained vectors with the 515/806 inserts. Then, five white colonies were selected from the plate containing E. coli cells with the vectors that had the NS1/NS2 inserts. Each of the colonies chosen were inoculated into a tube of 100µL of nuclease free water. Then 24 PCR tubes were taken and labeled 1-24. 7.5µL of nuclease free water was added to each tube, and then 12.5µL of the Taq master mix was carefully added to each tube. Following the Taq master mix, 2.5µL of the M13 F/R was added to each tube. Then 3µL of the inoculated E. coli cells were appropriately added to specific tubes. Colonies grown on the plate containing the negative control were added to tubes 1-3. The colonies that were present on the positive control plate were added to tubes 4-9 and the colonies that contained the 533F/1492R inserts were added to tubes 10-14. For tubes 15-18, the colonies that contained 515F/806R were added and for tubes 19-24, the colonies containing the NS1/NS2 inserts were added. Then each tube was run through a Thermocylcer. For the initial denaturing, the temperature was increased to 95ᵒC for 5 minutes. It remains at 95ᵒC for another 45 seconds and then the temperature is decreased to 50ᵒC for 1 minute for the primers to anneal. After 1 minute, the temperature is raised to 72ᵒC for the extension of the DNA for one minute and then the temperature is raised to 95ᵒC once again for 45 seconds. The cycle repeats 34 times. After 34 cycles, the temperature is raised to 72ᵒC once more for 10 minutes for the final extension. The samples are then stored at 4ᵒC. To measure the concentration of the DNA, Each sample was run through a Thermo Scientific Nanodrop 2000 Spectrophotometer.
DNA Mini-Prep:
Eight microcentrifuge tubes were labeled as MC1-MC8. 1 ml of culture was added to the tubes which were kept on ice at 4 degrees Celsius. The remainder was centrifuged at 14,000x g for ten minutes. The bacteria was re-suspended by adding 250 µl of the R3 solution and mixing it until the solution became homogeneous. 250 µl of the solutions labeled as L7 was added to the solution and it was mixed by inverting the solution using a micropipette. Then it was incubated at room temperature for five minutes. 350 µL of the solution labeled as N4 was then added to the microcentrifuge tube. The tube was then centrifuged at 14,000x g for ten minutes. After ten minutes, all of the supernatant was loaded onto a spin column which was placed in a wash tube. The column was centrifuged at 12,000x g for one minute. Then the flow through that was collected in the wash tube was discarded. 500 µL of W10 was then added to the column, and the column was incubated for one minute at room temperature. The column was then centrifuged at 12,000x g again for one minute. After centrifuging, the flow through in the wash tube was discarded. Then 700µL of the solution labeled as W9 was added to the column. The column was then centrifuged at 12,000x g for one minute twice and each time, the flow through was discarded. Then, the spin column was placed in a recovery tube and the wash tube was thrown away. 75µL of the solution labeled as TE was added directly to the center of the column and incubate for one minute at room temperature. Then centrifuge at 12,000x g for two minutes. Throw away the column. The flow through in the recovery tube should contain pure DNA. To find the concentration of the DNA, the DNA was run through a Thermo Scientific Nanodrop 2000 Spectrophotometer.