Ligation and Transformation
Ligation:
The pGEM-T vectors and the control insert DNA tubes were centrifuged to collect the contents at the bottom of the tube. 5 different 1.7 mL tubes were labeled according to what DNA insert was going to be added to each tube. The first tube was labeled as a negative control, the second tube as the positive control. The third tube was labeled as 533F/1492R, the fourth tube was labeled as 515F/806R and the fifth tube was labeled as NS1/NS2. 5µL of the rapid lagation buffer, which contained the T4 DNA ligase, was added to each tube. Then 1µL of the pGEM-T vector was added to each tube. Then, 2µL of the 533F/1492R PCR product was added to the third tube. 2µL of the 515/806 PCR product was added to the fourth tube and another 2µL of the NS1/NS2 PCR product was added to the fifth tube. Then, 2µL of the control insert was added to the second tube. 1µL of the T4 DNA Ligase was added to each tube. 3µL of water was added to the first tube, then for tubes 2-5, 1µL of water was added and mixed. The reactions in each tube were mix by pipetting. Each of the reactions were then incubated for one hour at room temperature.
Transformation:
Lucigen E. cloni: reba says mention that this is a sample or something so I'm not left wondering what it means5µL of the ligation reaction was added to five tubes. Then, 25µL of competent Lucigen E. cloni cells were added to each tube and were mixed with the ligation reactions in each tube. The tubes were kept on ice for five minutes. After five minutes, each tube was heated at 42 degrees Celsius for exactly 45 seconds. After 45 seconds, the tubes were removed and set back on ice for one minute. Then, 500µL of recovery media was added to each tube. Then, the tubes were incubated at 37 degrees Celsius for 45 minutes to one hour.
NEB 5-alpha Competent E. coli:
The same procedure that used with the Lucigen E. cloni was also used for the transformation of NEB 5-alpha Competent E. coli. However, the ligation reaction that was used for this transformation process contained newer reagent for higher quality.
One Shot TOP10 Chemically Competent E. coli:
The same procedure for transformation was followed again using the One Shot TOP10 chemically competent E. coli; however, the ligation reaction that was used in this transformation process only used the older reagents that were originally used for cloning of the Lucigen E. cloni.