DNA/RNA extractions
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What tissue is the easiest to work with
muscle
what will be needed to analyze plant cells?
lysis buffers for membranes and mixer mills to get rid of the cell walls
what is in cells?
RNA, DNA, proteins, small ions, sugars, fats, nucleic acids
when extracting DNA what is a bad sign?
colour
what is an inhibitor that we must always be mindful of?
calcium ions
in bones, teeth and tusks what are inhibitors and how do we get DNA?
Ca2+, melt down the bone until it becomes floppy
where do we sample on a feather/what types of feathers?
bottom we want either growing quills or blood quills
why is blood not a good sample?
because of all the +2 ions in the sample
what is the best place to extract DNA on a shell?
the inner membrane will give species ID
what do fecal samples tell you?
diet and sometimes mucosal coat telling you what/who deposited it
why is urine not very good?
most cells are absorbed into the ground/not deposited at all
how do you deal with bark, leaves, and seeds?
grind them up in mixer mills with metal beads
how many cells are needed for a full mtDNA profile?
1
how many cells are needed for a full nDNA profile?
167
how many pg of DNA are needed for a full profile?
500pg
What are good preservation buffers?
RNA later or lysis buffer
what is the best way to preserve blood?
FTA cards
what are sample preservation methods in the field?
• freezing • buffers (RNA later, or Lysis buffer) • EtOH • adding tissue to desiccant • FTA cards
what is the worst tissue choice in degraded tissues?
organs: too many enzymes and quick autolysis
where is the best DNA on dried/desiccated tissues?
ear tips, edges of pelts
if the tissue is clearly rotten what will be in higher amounts? where should you take DNA from?
bacterial DNA. plucked hairs, feathers, drilled out pulp of teeth or marrow
what is the correct concentration of DNA for PCR?
250pg/uL
Why is DNA negatively charged?
because of the phosphate sugar backbone tightly complexed with positively charged histones
what is the first step in DNA extraction?
dissolve tissue in buffer: breaks cell/nuclear membranes, disrupts DNA -protein complexes, binds multivalent ions