DNA/RNA extractions Flashcards
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
During step one of DNA extraction what solutions are needed?
- Urea
- n-lauroyl sarcosine (or SDS)
- CDTA (or EDTA)
what does n-lauroyl sarcosine do?
binds proteins and unfolds them while long hydrophobic aliphatic chains bind to hydrophobic backbones of DNA the charged group makes complex soluble in aqueous buffers
what does CDTA do?
Chelating agent binding multivalent ions (Mg and Fe)
what is step 2 of the DNA extraction?
add proteinase-K to digest proteins
What happens to cells lysed with DTT?
breaks protein disulfide bridges prevalent in hair, nails, horns, claws, antlers, and other structures high in keratin content
what is an extra step for materials like bone, teeth and ivory? and why?
soaking in EDTA to decalcify prior to extraction
what is step 3 in the DNA extraction?
separating DNA from digested proteins
how is DNA separated from digested proteins
- organic solvents
- silica to bind DNA
- magnetic beads bind to DNA
In step 3 what was the traditional method of DNA separation?
adding organic solvents such as phenol and chloroform to precipitate and denature the proteins
When cell lysate is treated with buffer saturated phenol and chloroform what happens?
DNA remains in the aqueous phase while cellular proteins extracted into organic phase
When do nucleid acids bind to silica?
in the presence of chaotropic salts
Iron from RBC is a PCR inhibitor how is this fixed?
FTA; ACK, and centrifugation
dilution is a solution for what inhibitor(s)?
melanin (hair), and proteins (not successfully removed in extraction)
ethanol is a PCR inhibitor how is this fixed?
heat elution with lid off
minerals from bone is a PCR inhibitor how is this fixed?
decalcification prior to extraction
what is swabbed on a fecal sample?
mucosal coat because there are fewer inhibitors
what are the best fecal samples to collect?
fresh or on snow
why is species-specific quantification important?
- estimate efficieny of extraction method; various methods yield different amounts and quality of DNA
- it is especially important to dilute samples to known concentration for use in PCR, conserves DNA, and reduces the possibility of introducing inhibitors which may be present in the extracted solution
What is phenol?
an organic solvent that removes proteins it is a benzene ring with an OH on it
what does the ethanol step do during phenol-chloroform extraction?
dehydrates the DNA and makes it precipitate
how do you rehydrate the pellet?
add in 70% ethanol
why do you need to get rid of all the ethanol during a phenol-chloroform extraction?
because it is a PCR inhibitor
T/F: ethanol prevents DNA from dissociating
False, you will always lose some DNA during this protocol.
why do you add a weak solution of EDTA in the last step?
because it is a preservative
why might an aqueous phase be slightly red?
caused by some iron in the solution.
what is a chaotropic salt?
what do the magnetic beads do?
prequantify how much DNA you might get
why do you have a negative and positive control?
negative says if reagents are contaminated (at the start), positive evaluates efficiency of extraction (at the end)
in negative if there is any DNA amplification, is this bad?
not the case FSC will accept 1:10 ratio since its kinda impossible to get 100% clean
why is melanin hair dilution kinda bad?
because its not super concentrated so it might just dilute out the small amount of DNA in the hair
