Supplemental File S4: Additional assessment exercises that were not included in the original
activity.
Learning Objectives
Assessment Exercises
Demonstrate which parts of the
Instructor:
primers will anneal to the original Create a worksheet showing a double-stranded DNA template
DNA template.
sequence (50-75 bp), specific oligonucleotide primers modified
with 5’-end localized EcoRI restriction enzyme sites, and a plasmid
Demonstrate how the
vector with an EcoRI site included in the multiple cloning site. The
incorporation of primer restriction worksheet could also include one or more restriction enzyme
enzyme sites into PCR products is recognition sequences, including EcoRI (Supplemental File S6).
a useful tool for subsequent
cloning of the product into a
Students:
vector.
Locate the primer annealing sites on the template DNA and indicate
which parts of the primer will anneal.
Draw the products of a PCR reaction (after 30 cycles).
Locate and clearly mark all of the sites on the PCR products that
would be recognized by EcoRI.
Draw the resulting recombinant vector(s) after successfully cloning
of the PCR product.
Notes:
1) Instructors may or may not choose to include an EcoRI site in
the DNA template sequence. Including the EcoRI site in the DNA
template sequence would highlight that modifying the primers with
5’-end EcoRI sequences would not be ideal for cloning this DNA
fragment.
2) Students should draw two possible recombinant vectors since the
PCR product could be cloned in either direction due to the use of a
single restriction enzyme site on both primers.
3) This exercise could be expanded to include interpretation of
DNA band sizes after electrophoresis and troubleshooting of PCR
concatamers ligated into a single vector.
Calculate the length of PCR
products.
Instructor:
Create a worksheet showing a double-stranded DNA template
sequence (50-75 bp) and a variety of oligonucleotide primers with
different 5’- and 3’-end polarities (Supplemental File S6).
Students:
Predict which primer pairs will produce a double-stranded DNA
product.
Determine the size of the DNA product(s).