what are the 4 steps of DNA isolation?
- lyse: break apart
- bind: DNA to column
- wash: contaminants away
- elute: DNA from column
how does lysis work?
a lysis buffer containing detergent disrupts the membranes and denaturants to release DNA
how does purification (wash) work?
uses buffer solutions and ethanol to remove contaminants e.g lipids, proteins
how does elution work?
contains an elution buffer that allows DNA to release itself from the column and is eluted into the tube at the bottom
what is the wavelength for the isolation of DNA and the lamda max
260nm/280nm ratio
LM: 260
what are the steps of PCR?
- denaturation
- annealing
- extension
what does emolgenin tell us in DNA profiling?
sex
what does it mean if there is one peak or two peaks at particular positions on the gene?
one peak (homozygous)- inherited one gene from both parents
two peaks (heterozygous)= inherited one gene from each parent
what is the allelic ladder?
shows what the kit looks for in the test (references). has a mixture of known fragment sizes that are used to determine sizes of unknown DNA fragments
what are the rules regarding peak height imbalance
they must be within 50% of each other to be an allelic pair
what are spikes indicative of?
instrumental problem
what would you typically see on a degraded DNA profile?
a distinctive slope
what are the purpose of the denaturation step?
samples are heated to 95 degrees to separate the target DNA into single strands
what is the purpose of the anealing stage in PCR?
temperature is lowered to 65 degrees to allow the left and right primers to anneal to their complimentary sequences
what is the purpose of the extension step in PCR?
temperature is raised to 72 degrres allowing taq polymerase to bind at each primes site and extend a new DNA strand
what does taq polymerase do during PCR?
takes on side of DNAs double helix structure and duplicate it making a new DNA strand
