Objectives – Lab Materials
Nested PCR
1. You should understand the fundamental concepts behind PCR. How does nested PCR differ from
“regular” PCR? Why would one use nested PCR?
2. What is the purpose of treating our initial PCR reaction with exonuclease I? What was the
purpose of the 80°C incubation following exonuclease I treatment? Is this step really necessary?
Analysis and Purification
1. What is the theory behind size exclusion chromatography? Why did we need to purify the PCR
fragment?
Ligation and Transformation
1. What is the purpose of cloning the nested PCR fragment?
2. Be sure to understand why each step of the protocol was performed.
a) What is the purpose of adding the proofreading polymerase to our purified fragments?
b) What is the purpose of adding ligase to your cloning reaction?
c) What is the purpose of the heat shock?
d) How can the researcher differentiate between true transformants, recircularized vectors,
circularized inserts and bacteria that were not transformed?