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scientific discovery
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P20
measures 2 to 20 uL
P 100
used to measured 20 - 100 uL
P 1000
used tp measure 100 to 1000 uL
Post rinsing
used when pipetting less then 20 uL, ensures that all liquid in micropipette is removed
when is a pipette most accurate?
use the smallest possible pipette for volume transfer
micropipettes are most accurate in the middle to top end of their volume range
preventing contamination in the lab
wipe the stuff down
wear and change gloves often
open samples carefully and keep closed whenever often
use barrier pipette tips
change pipette tips after use
use sterile equipment
crude prep
rapid DNA extraction yielding a solution containing DNA that is used in PCR and some residual cell components and RNA
abrasion and physical destruction of cell wall and nucleus (cell things)
detergent and salt are used to dissolve and dissociate proteins
centrifuge to remove tissue fragments
Isopropanol
centrifuge
ethanol
centrifuge
machine designed to separate heavier materials from lighter materials within a substance
pellet
heavier particles that clump at the bottom of the tube after being centrifuged
supernatant
solution (lighter particles) above the pellet
why do you angle the hinge towards the center of the centrifuge?
to ensure the pellet forms directly below the hinge of the microcentrifuge tube
ethanol
washes impurities out of the DNA pellet without dissolving it
isopropanol
precipitates (separates) DNA turning it into little piece’s
DNA template
comes from organism of interest contains DNA sequence that will be analyzed
PCR primers
short pieces of single-stranded DNA with specific sequences.
Forward and reverse PCR primers
one binds upstream target DNA helix one binds down stream
two primers are needed
how do PCR primers bind to DNA polymerase?
Provide a 3’-OH group which DNA polymerase needs to begin synthesizing a new template strand
DNA (Taq) polymerase
enzyme that synthesizes new DNA strand
heat stable enzyme which is typically used in PCR
links dNTPs together based on the sequence of the DNA template
Deoxyribonucleotide Triphosphates (dNTP’s)
the four monomer units from which a new strand of DNA.
PCR reactants/ Master mix
DNA polymerase
dNTPs
buffers and salts
barcoding regions
specific regions of DNA template that is amplified.
used to classify organisms
around 500-800 bp long
PCR thermal cycling steps
denaturation
primer annealing
extension
repetition 30-40 times
thermocycler/ PCR machine
machine that rapidly heats and cools across a range of extreme temperature differences
denaturation
sample is heated between 90-100*C. heating causes hydrogen bonds that hold double stranded DNA together causing the strands to separate
Annealing
rapid cooling which allows DNA primers to bind to the separated template strands
extension
DNA polymerase added DNTPs to the 3’ end of the primers making complete copies of each strand
DNA barcoding
using a short section of DNA from a specific gene to identify a species
why was mitochondrial DNA chosen?
the DNA sequence surrounding the variable region is basic enough that a non Specific PCR primer in needed
the variable region is divergent enough that you cand tell species apart (low intra-specific but high inter-specific divergence)
there are many identical copies of the target region
PCR amplicon
The DNA fragment produced by (PCR). It is a specific region of DNA that is amplified using primers and DNA polymerase.
agarose gel electrophoresis
a technique used to separate and view fragments of DNA based on molecular size
separates DNA fragments based of base pair size
a current is sent through a buffer covered gel
agarose gel slab
traps DNA fragments as they move through based on their bp size
the smaller the fragments the further they move through the slab ]
covered by buffer solution it is made with
how do DNA fragments move through gel
fragments placed in wells close to the cathode (negative end) and repulsed and dragged toward the anode (positive end)
DNA is polar and negatively charged therefore repulsed by the negative charge
TAE buffer
a conductive solution that agarose gel is made from and covered by during gel electrophoresis
molecular ladder
a sample containing DNA fragments of known sizes which is placed in the first well and used to trach the position of unknown (sample) DNA fragments
SYBR safe
dye that fluoresces under UV light when bound to DNA
works by slipping between base pairs and fluorescing
paraphrasing
expressing an author’s ideas in your words by changing both the language and the sentence structure
patchwriting
rewritten passage that is too close to the original text. considered plagiarism
quotation
copying an authors writing directly using citation
summarizing
putting the main ideas from another source into your own words and giving the author credit for the information
proteinase K
digest contaminating proteins during DNA extraction
vortex
mixes liquid