Protocols

[1A] Polymerase Chain Reaction

PCR is used to rapidly amplify certain segments of DNA that are selected via designed primers. This protocol was used to accomplish a variety of goals, from extraction of parts from a cloning vector to the addition of restriction cut sites at the ends of parts.

1. Defrost template, 2x Master Mix and primers
2. Determine desired total working volume (~25uL)
3. Label reaction tubes
4. Create below reaction mix
5. Place mixture in thermal cycler at settings shown below

Sample Reaction

This is an example protocol used in the thermal cycler, durations and temperatures subject to change

[1B] PCR Cleanup

The Qiagen Qiaquick PCR cleanup kit was used to purify the thermal cycler product and remove remaining polymerases and nucleotides.

1. Gather the solutions from the PCR cleanup kit
2. Mix 5 times volume of Buffer PB with PCR reaction tube
3. Add 10uL 3M sodium acetate
4. Spin down the mixture in a QIAquick filter column at max speed for 60s. Discard flow-through and replace column into tube
5. Add 750uL of Buffer PE to the mixture and spin down at max speed for 60s to wash. Discard flow-through and replace column
6. Centrifuge the tube one more time to remove any remaining wash buffer
7. Place column in clean 1.5mL plastic tube
8. Add 30uL of Buffer EB to the column and let stand for 1min. Centrifuge at max speed for 1 min.