BCMB 406B Midterm 1 study guide 2024

What is the most important factor in a successful PCR reaction? - Well designed primers What can the theory of primer design also be applied to? - The use of oligonucleotides in other techniques that utilize annealing or formations of duplexes of nucleotides (probes in Southern blot, Northern Blot, ect.) What variables influence the success of a PCR reaction? - - quality of template DNA - cycling conditions (melting temp., duration) - specificity and stability of the PCR primers What criteria must primers fill? - Must fulfill criteria independently and in combination: - Primer length - Percent GC content - Melting temperature - Sequence * Length, % GC, Tm = linked, in that modification in one will alter the others ** Sequence is dependent on the target sequence What will the target sequence of a primer determine? - - the first 3 criteria (length, % GC, Tm) - influences the likelihood that a primer will form hairpins and dimers with itself, form cross-dimers, with other primer - these are undesirable as they effectively decrease the amount of available primer to initiate the PCR reaction, and therefore gives less product. Part A Objective - To analyze two primer pairs and design primers for a given DNA sequence with Primer3 and Net Primer. Denaturation (PCR Conditions) - - PCR protocol starts here - 94 ºC - 1-5 mins, but length will depend on nature of the template DNA - Each cycle after is 30-45 sec -- Ex. Plasmid DNA is denatured in 1 min, 3-5 mins to denature chromosomal DNA