620 oral boards

amplification kit allows for ____________ amplification of CODIS and additional loci

simultaneous/multiplexing

what kit and instrument are used for amplification?

GlobalFiler kit and PCR Veriti Thermal Cycler

T/F: amplification reactions should be set up in a separate area from where extraction was set up

true

what is the recommended amount of DNA for amplification?

1.0 ng

what is the purpose of vortexing and centrifuging tubes?

accumulate contents at bottom of tube

general procedure for amplificaiton

mix/centrifuge samples. prepare mastermix. add mastermix, template DNA, water to wells (10uL mastermix, 15uL DNA/water). centrifuge. program thermal cycler. run and store samples

what are the components of the amplificaiton mastermix?

reaction mix and primer mix

what is included in the positive and negative controls for amplification?

positive control is control DNA and water. negative control is water

what are the settings (temperature, time, cycles) for the thermal cycler in amplifiaction?

95C for 60s, 94C for 10s and 59C for 90s for 29 cycles, 60C for 10min, 4C for infinity

T/F: samples are stored in the freezer after amplification

true

how are amplified STR loci detected and separated?

based on size and locus

what instrument is used for capillary electrophoresis?

3500 genetic analyzer

DNA fragments are separated through the application of a ________ charge (DNA is negatively charged). this charge draws the DNA through the polymer in the capillary

positive

T/F: smaller fragments navigate through the polymer more quickly than larger fragments

true

fragments are illuminated by a _____ at the detection point. this allows the attached dyes to fluoresce

laser

the attached dyes that are excited are in _______ that are unique to each locus

primers

what detects the amount of fluoresce emitted in CE?

camera

information from CE is used to construct _________________

electropherograms

general procedure for CE

make mastermix. determine layout of plate. add mastermix and sample/ladder or add formamide. set up to run samples. load samples. run data

what are the components of the CE mastermix?

formamide and LIZ 600 (internal lane standard)

T/F: formamide is added to empty wells of an unfilled column

true