Transcript PCR Techniques

PCR Techniques
Basics of PCR
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Primers
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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
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Denature
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Anneal
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Standard is 5C below Tm of primer (the portion that anneals to your gene)
Can do gradient on our thermocycler
Extend
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Can be extended in GC rich templates
68-72C
Polymerases
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TAQ—faster and better at amplifying long genes
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PFU—slow but provides proofreading
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Adds a A to sequence
Error prone
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 510C 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?