BCMB 406B - Lab 1 exam questions and solutions

The success of a PCR reaction is mostly dependent on what? - well designed primers Primer design can also be applied to what? - - probes in Southern and Northern blots - DNA sequencing - microarray design What else can affect the success of a PCR reaction? - - Quality of the template DNA - cycling conditions - Specificity and stability of the PCR primers What are key criteria in primer design? - - Primer length - Percent GC content - Melting temperature - Sequence How are Primer length, Percent GC content and Melting temperature linked? - in that modifications to one will alter others Target sequence determines what? - - It will determine primer length, percent GC content and melting temperature. - influence the likelihood that a primer will for hairpins and dimers with it self or form cross dimers What was the purpose of part A of lab 1 procedure? - To analyze two primer pairs and then designing our own primers using Primer3 and NetPrimer Standard denaturation temperature is what? - 94 degrees celsius Taq polymerase and phusion polymerase function best at what temperature? - 72 degrees celsius A PCR protocol initially starts with what? - Initial denaturation for 1-5 minutes at 94 degrees celsius -- length of time depends on the template DNA Denaturation times for template DNA - - Plasmid: 1 min - Chromosomal DNA: 3-5 min Denaturation takes how much time? - Its the most consistent between 30-45 seconds Annealing takes how much time? - Generally about 30 seconds Elongation time is dependent on what? - - On the length of the product to be produced, the longer the product the longer the elongation time, and on the polymerase used - About 30 seconds for 1000bp