bio lab exam

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

1. abrasion and physical destruction of cell wall and nucleus (cell things)

2. detergent and salt are used to dissolve and dissociate proteins

3. centrifuge to remove tissue fragments

4. Isopropanol

5. centrifuge

6. 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

1. denaturation

2. primer annealing

3. 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