Presentation on theme: "PCR Techniques. Basics of PCR Primers –15-60bp (60bp is limit synthesized by IDT) –Annealing temp ideally >55C (portion that anneals to your template)"— Presentation transcript:
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 –TAQfaster and better at amplifying long genes Adds a A to sequence Error prone –PFUslow but provides proofreading Used for mutagenesis primarily –TAQ:PFU mixqualities of both
Touchdown PCRcan 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 dont 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 weve 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 doesnt work, and sew together –Setup reaction with very small (<0.1ul) of middle primers and 1ul of outer primers) –Normal PCR
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?