1 / 15

PCR Techniques

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

chiquita-clayton
Download Presentation

PCR Techniques

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. PCR Techniques

  2. 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

  3. 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)

  4. 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

  5. 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

  6. 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

  7. 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)

  8. 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/

  9. Example 1 • You receive a vector containing cDNA for protein X in a T7 expression vector---you want it His/TEV tagged • Options?

  10. Example 2 • You are expressing a protein in yeast via a vector and want to change the promoter type for overexpression • Options?

More Related