PCR Techniques

Basics of PCR• Primers

– 15-60bp (60bp is limit synthesized by IDT)– Annealing temp ideally >55C (portion that anneals to your template)– Hairpins Tm<50 ?– Self dimers---only important if they are 3’ annealing dimers– Silent mutants---better to have them on 5’ end than on 3’ end

• Cycles– Denature

• Can be extended in GC rich templates– Anneal

• Standard is 5C below Tm of primer (the portion that anneals to your gene)• Can do gradient on our thermocycler

– Extend• 68-72C

• Polymerases– TAQ—faster and better at amplifying long genes

• Adds a A to sequence• Error prone

– PFU—slow but provides proofreading• Used for mutagenesis primarily

– TAQ:PFU mix—qualities of both

Touchdown PCR—can be applied to mutagenesis

• Start with annealing temp greater than or equal to your primer Tm (I usually do 2C over)

• Decrease annealing temp each cycle– 1C or 0.5C

• Continue decreasing until annealing temp is 5-10C below Tm

• For remaining cycles maintain low annealing temp or go back up (if your primer has 5’ extensions)

Basics of mutagenesis

• 20-25bp flanking mutation

• Linear amplification

• Primers need to anneal to each other with reasonable Tm (>50C ?) to allow E. coli to fix plasmid

Long insertions/mutations

• Primers don’t have to be complements of one another

• 3’ end anneals to template

• 5’ end is insertion (allows for up to 40bp insertion)

• For reverse primer: 5’ end is complement of insertion and 3’ end anneals to other side of insertion site

Megaprimer mutagenesis

• What if you want to insert >40bp and up to 2kb (largest we’ve successfully done)

• 1. Do PCR of your gene – 5’ extensions on your primers that are

complementary to your vector

• 2. Gel Extraction

• 3. Do mutagenesis reaction– Substitute 500ng PCR product for primers

Fusion PCR

• What if you want to join two genes together (or a promoter with a gene)

• Can use megaprimer mutagenesis and clone genes in one at a time

• 1. Design internal primers that have 5’ overhangs that are complementary to fusion gene (Tm>55)

• 2. Do PCR of each gene individually• 3. Do gel extraction kit• 4. Add small amount (0.1ul) of each pcr product into

another PCR reaction with only the forward primer of the first gene and the reverse primer of the second gene

• 5. Do normal pcr with this mixture (make sure to account for the Tm of the overlap region)

Gene synthesis

• Overlaping primers (3’ overlaps)– Analogous to fusion pcr– Can synthesize fragments if whole doesn’t

work, and sew together– Setup reaction with very small (<0.1ul) of

middle primers and 1ul of outer primers)– Normal PCR

• http://genomes.urv.es/OPTIMIZER/

Example 1

• You receive a vector containing cDNA for protein X in a T7 expression vector---you want it His/TEV tagged

• Options?

Example 2

• You are expressing a protein in yeast via a vector and want to change the promoter type for overexpression

• Options?