Exam 1 Flashcards
define plasmid
extrachromosomal, double stranded, usually circular, supercoiled DNA molecules. found in many bacterial species independent of chromosome
how to balance centrifuge
center of mass must be in the middle of the rotor. tubes should all be the same mass (same volume). place tubes evenly around the centrifuge.
even numbers place opposite each other in groups of two. odd number, place 3 in an even triangle
only numbers that CANT be balanced are 1 and 1 less than the number of spaces.
standard lab plasticware
microcentrifuge tubes, 0.5, 1.5, and 2 ml
PCR tubes, 0.2 ml
96 well plates, standard 8x12 layout
general types of pipettes
automatic pipettes transfer small liquid volumes accurately
glass pipettes are not accurate for volumes <1 ml
continuously adjustable digital pipettes, each can be set to transfer any volume within its own volume range
for optimal reproducibility in pipetting
- Consistent speed and smoothness when pressing/releasing plunger
- Consistent pressure on plunger at first stop
- Consisten and sufficient immersion depth
- Nearly vertical positioning of pipette
- Avoid all air bubbles
- Never lay the pipette on its side or invert the pipette if liquid is in the tip
types of pipette tips (attachment systems)
Conical: oldest, most common, shaft wedges into tip held on by friction
LTS: less common, more expensive, cylindrical shaft has better seal and low ejection force
ClipTip: proprietary but not much more expensive, tips firmly attach with better seal and low ejection force
explain filter tips
pipettes can be contaminated by aerosols from the sample. filtered tips prevent contamination and are a good idea when low levels of contamination may be a problem (eg PCR)
pipette tip sizes
2.5 and 10 (red)
20, 100, and 200 (yellow)
1000 (blue)
what are multichannel pipettes
pipettes with a row of tips. good for high throughput work
what are positive displacement pipettes
piston is integrated into the tip and there is no dead air space and so no contamination issues. they are more accurate and have no issues with capillary action. they are not very common
(typical pipette is an air displacement pipette)
state the nucleic acids
Purines: Adenine and Guanine
Pyrimidines: Cytosine, Thymine, Uracil
Deoxyribose refers to the lack of OH on C2
Ribose has an OH on C2
naming of free bases vs a ribose attached base
Free Base: -ine. Adenine, guanine, cytocine, thymine
attached base w/ no phosphate (ribonucleoside). Adenosine, guanosine, cytidine, thymidine
attached base w/ phosphate(ribonucleotide). adenylate, guanylate, cytidylate, thymidylate
how are nucleic acids linked
phosphodiester bond between 3’ carbon and 5’ phosphate
why do As match with Ts and Gs with Cs?
H bonding between the structures is complimentary. Purines (2 ringed) match with pyrimidines (1 ringed) to make a 3 ringed structure
A and T make 2 H bonds
G and C make 3 H bonds
2 main differences between DNA and RNA
uracil (RNA) and thymine (DNA)
2’OH on ribose (RNA) and 2’H on deoxyribose (DNA)
factors that affect complimentary strands interacting
complimentary strands form by hybridization (annealing) and this can be increased with high salt and low temperature.
Opposite is denaturation (melting) and can be increased with low salt and high temperature
review the important unit conversions
milli - 10^-3
micro - 10^-6
nano - 10^-9
pico - 10^-12
average weight of a DNA basepair
650 daltons
daltons is g/mol
how to quantify DNA via absorbance of UV light
aromatic bases have absorbance maximum at around 260 nanometers.
1.0 A260 = DNA concentration of 50 micrograms per ml (double stranded DNA) or 38 micrograms per ml (single stranded DNA/RNA)
instruments that quantify DNA
spectrophotometer and nanodrop
works by a fiber optic cable in measurement arm
conditions of DNA quantification via UV absorption
effective range is narrow: A260 from 0.05 to 2.0, which is DNA concentrations from 2.5 to 100 micrograms/ml
sample must be very pure for accurate measurements as stuff can absorb at 260 nm (RNA, EDTA, phenol
how can concentration be determined by absorbance?
DNA has molar extinction coefficient, use in Beer-Lambert law: I = Io10-Edc (just look it up). amount of light that gets through depends on what’s in it and how much of it there is
Basically multiply A260 by 50 because 1 A = 50 micrograms/ml DNA
what is A260/A280
nucleic acids absorb at 260 and proteins at 280. the ratio of absorbances at these wavelengths is used as a measure of purity. ratio of 1.8 is accepted as pure for DNA and ratio of 2 is pure for RNA. low values indicate protein contamination
what do abnormal high or low 260/280 ratios indicate?
usually indicate sample is contaminated by protein or reagent such as phenol or an issue with measurement.
low ratio is caused by residual phenol/other extraction reagent or a low concentration
high ratio is not an issue
what is A230 and 260/230 ratios
the result of other contamination, generally organic solvents. 260/230 ratios in range of 2.0-2.2 are pure for nucleic acids
why is contamination a problem in DNA quantification
may result in an overestimation of the nucleic acid concentration and negatively influence downstream analysis
how to identify contamination in DNA quantification
look at 260/230 ratio: a low ratio may be result of contaminant absorbing at 230 nm
look at 260/280 ratio: a low ratio may be result of contaminant absorbing at 280 nm
wavelength of the trough in sample spectrum should be at 230 nm
wavelength of the peak in sample spectrum should be 260 nm
what do abnormal high or low 260/230 ratios indicate?
low 260/230 ratios may be result of: carbohydrate carryover, residual phenol, residual guanidine, glycogen
high 260/230 ratio may be result of making a blank measurement on a dirty pedestal, using an inappropriate solution for the blank measurement (one that is not similar to the sample solution)
describe quantification in practice
measure concentration after cleanup. blank the spectrophotometer using the same solution the DNA is in. measure OD at 230, 260, and 280 nm. Use OD260 to calculate concentration and use ratios to asses purity
describe fluorometry
protein and nucleic acids can be quantified using a fluorescent dye which binds. A higher emission correlates to higher concentration. it is more sensitive, effective over a wider range, but more labor intensive and expensive.
The dyes are SPECIFIC and so do not show contaminations or other molecules
what are restriction endonucleases
enzymes which cleave within the DNA strand at a specific site. they work by hydrating the phosphodiester bond and then the H bonds are not sufficient to hold the strands together
How are REs site specific
They cut on axis of symmetry, most often a palindrome
types of REs
Type I: cuts a distant site, not very useful or important
Type II: cuts in/near recognition site. This is the common and useful type
Type III: non palindromic, not super useful
Type IV: recognizes modified DNA
Type V: uses RNA guide (CRISPR)
explain sticky vs blunt ends
sticky ends have an overhand that can be used to pair with another compatible strand. blunt is just a solid cut off, no overhangs
how often will a RE cut DNA molecule? average fragment length if you toss a RE at an unknown DNA sequence
depends on the length of the cutter. 4 to the power of the length, eg a 4 bp cutter will cut every 256 base pairs and average fragment length would be 256.
But sequences are not distributed randomly so this is unlikely to actually be true. Use a restriction mapper on a known sequence to see how many cut sites there are
ingredients in restriction digest
Pure water Buffer - control pH and contain salt DNA that you want to cut Bovine serum albumin - stabilizing agent Restriction Enzyme - stored in glycerol to preserve/stabilize. should not exceed 10% of total rxn volume
How much DNA will 1 unit of RE digest in 1 hour
1 unit of RE will digest 1 µg of DNA in a 50 µL reaction in 1 hour.
can use enzyme:DNA:reaction volume ratio when designing reaction. Typical conditions are a 10 fold overdigestion so like 10 Units of RE and 1 µg DNA
how do you stop a restriction digest reaction
use a buffer containing EDTA which chelates cations needed by enzyme for the reaction
heat inactivate enzyme by raising to 65 or 85 C
use phenol/choroform extraction or column clean up, precipitates
what is a double digest
cleaving DNA with two different enzymes at the same time. careful which combinations you use, some are not recommended. use buffer that has most activity for each enzyme
what is star activity
relaxed or altered specificity, the RE cuts outside of its usual sequence
what causes star activity
high glycerol concentration, >5%
high enzyme to DNA ratio, >100 U/µg
low ionic strength (<25 mM) or high pH (>8.0) buffer
prolonged reaction time
presence of organic solvents (DMSO, ethanol, etc)
substitution of Mg with other divalent cations
what is the point/usefulness of restriction digests
cloning: cut pieces to put back together in different combinations
diagnostic information: determine direction of insert, order of fragments, presence of mutations
general features of ALL vectors
origin of replication
multiple cloning site
selection marker
what is the replicon
governs replication of plasmid and number of plasmid copies per cell. includes origin of replication and association factors
most common replicon used is modified pMB1, maintains about 500-700 copy number
what’s the deal with copy number? high vs low
high copy number plasmids are the workhorses of molecular cloning. used for almost all routine manipulation of small recombinant DNA (<15 kb)
low copy number plasmids are for genes that are lethal or unstable in high plasmids. also used for constructing Bacterial Artificial Chromosomes (BACs) that propagate large DNAs (>100 kb)
describe features of general cloning vectors
MCS is usually very extensive to help with subcloning
Often has gene to screen for recombination (lacZ-a for blue-white screening)
RNA polymerase promoters on either side of MCS to make RNA transcripts
Standard primer sequences on either side of MCS for sequencing of cloned sequence
features of vectors for gene expression
Promoter upstream of MCS for expression in desired system (constitutive promoters work in any cell)
Poly-A site downstream of MCS to add the poly A tail
May have mammalian selection marker for generation of stable transfectants
Also make sure to have 5’UTR and 3’UTR and start/stop codons included
what are fusion proteins? why make them?
fusion proteins are made from expression vectors that are designed with N terminal or C terminal fusions. tags can be used for detection, purification, fluorescence (to see location), and to make novel function proteins
what is IRES
internal ribosome entry site (IRES) is used for simultaneous expression of two genes. it makes bicistronic mRNA
what are inducible promoters
inducible promoter systems regulate timing and/or level of expression in a vector. generally, transcription factor driving expression of gene is either active or inactive in presence of specific chemical. chemicals used include tetracycline, ecdysone, mifepristone
what are reporter vectors
used for studying transcription. promoter or enhancer is cloned into MCS to drive expression of reporter gene. the gene produces a measurable/quantifiable product to analyze activity of regulatory elements in different conditions. common reporter genes are luciferase (measure luminescence), GFP or dsRed (measure fluorescence), and B-galactosidase or SEAP or CAT (measure enzyme activity)
describe how reporter vectors are opitmized
often optimized to climate as many transcription factor binding sites as possible.
poly-A site directly upstream of MCS to terminate any transcription that may start upstream of cloned promoter.
reporter may be destabilized to shorten half life so that the gene product with reach steady state level faster (eliminates differences in timing) or so that repression of a promoter may be studied
describe phage vectors
bacterial virus, linear genome.
37-53 kb
usually used for genomic or cDNA libraries
describe cosmids and fosmids
cosmids are plasmids with cos sites, allows phage particle packaging. can carry 45 kb
fosmids are similar but are based on the f-episome instead of lambda phage
describe BACs, YACs, and HACs
bacterial artificial chromosome: large plasmid can carry 350 kb
yeast artificial chromosome: linear, will replicate in yeast cells, can carry 1 Mb
human artificial chromosome: for use in mammalian cells. allows creation of transgenic cells
describe virus vectors
adenoviruses, lentiviruses, retroviruses. derived from pathogenic viruses modified for research. enable high transduction efficiency (good at getting DNA into high percentage of cells)
why make clones?
study protein function make protein with novel function study regulatory elements targeting vector for mutagenesis make a probe library of DNA molecules more
general steps in designing clone
- think carefully about what the clone needs to do
- figure our what parts are needed and in what configuration to do that purpose
- find a vector
- if some needed parts are not available, figure out how to get them (PCR, gene synthesis, etc)
- devise a strategy to build the final construct you need. Consider: orientation, reading frame, restriction enzyme sites
how to make RNA probe
clone is cut on one side of the insert and transcribed from the other end. probe needed for hybridization (blotting)
important points about promoter clones
orientation is important. make sure insert is pointing the right way
how much of the promoter do you need? (depends on the gene)
generally, 3’ end of promoter fragment should be between the transcription initiation site and the start codon
always check that your RE sites are not present inside the insert
important points about fusion proteins
must get the reading frame correct
check for stop codons. remove the stop codon of the original gene if making a C terminal fusion or the translation will stop before the fusion
do you know how to check that reading frame is going to be correct?
lecture cloning strategies
slide 14
how to fix a reading frame problem
find different RE sites to use
digest with one RE or both, then blunt the ends with nuclease or polymerase
design PCR primers to yield desired reading frame - add/subtract nucleotides from 5’ ends of primers, add RE sites to primers to facilitate cloning
use a different version of the vector. many expression vectors come in three versions, one in each reading frame
what is bioinformatics
combines computer science, biology, statistics, and math. uses software tools to analyze biological data. becoming very important as large datasets (like DNA sequences) cannot be analyzed by hand
examples of bioinformatics uses
sequence analysis:
- genome annotation, marking genes and other features in a genome
- comparative genomics, analysis of genomic structures among species to study evolution events
- genetics of disease, large scale genomic sequencing of many individuals makes it possible to search for genetic causes of disease
gene expression:
- microarrays and next generation sequencing can analyze expression of ALL genes in a sample
literature
- search and correlate many publications
NCBI
national center for biotechnology information
first place to start when looking for data. has the data and the analysis tools. genomic, protein, SNP, phenotype, homology, structure, and more
GenBank
NIH genetic sequence database of ALL publicly available nucleotide sequence and their protein translations.
RefSeq are reference sequences: non-redundant sequences as a baseline reference for genes proteins etc. 2 letter prefixes indicate RefSeq - NM mRNA, NP protein, XM or XP predicted, NC or NG or NT part of genome
BLAST
basic local alignment tool
aligns input sequence (nucleotide or peptide) with specified database. can translate to compare nucleotides to protein databases and vice versa.
Gene database
collects and integrates data from a wide range of databases and other sources. includes info about: nomenclature, reference sequences, maps, pathways, variations, phenotypes, other resources
Genome
whole genome sequence and annotation for thousands of species. displayed graphically, shows genes, repeat regions, regulatory region, and other features
PubMed
database of biomedical literature
GEO
gene expression omnibus
database of array and sequence based gene expression data. has tools to search for genes to look for expression information, good start to avoid repeating work
UCSC browser
search gem one to find gene of interest in genomic context. shows different types of data in tracks: genes, SNPs, mRNAs, DNase hypersensitive sites, alignments with other genomes, etc
Galaxy
graphical interface for many of the common tools. web based and configurable, can install new tools. used for true bioinformatics
what is PCR
polymerase chain reaction. used to amplify a single or few copies of a specific sequence of DNA exponentially
basic process of PCR
- denaturation - 98 C for 1-10 min. denatures DNA into single strands and activates polymerase in Hot Start PCR
- annealing - 50-65 C for 20-40 sec. temperature must be optimized for primers. primers bond with complimentary template sequence and polymerase binds to begin DNA synthesis
- extension/elongation - 72 C for 30-90 sec. time depends on length of DNA fragment and polymerase type. usually about 1000 bases per minute.
- repeat 1-3 about 25-40 cycles.
- Final elongation - 72 C for 5-15 min. ensures all single stranded DNA is fully extended
- final hold - 4 C forever. for short term storage
why do PCR
to obtain material for cloning or sequencing or in vitro studies
to verify identity of engineered DNA constructs
to monitor gene expression
to diagnose a genetic disease
to reveal the presence or identity of a micro organism
to identify an individual
what you need for PCR
Template DNA Primers Thermostable DNA polymerase Buffer, dNTPs, MgCl2 Thermocycler
basic rules of primer design
18-22 bp length: long enough for specificity but short enough to easily bind at temp
55-64 C melting temp: too high have tendency for secondary annealing. Tm = 4x(G and C) + 2x(A and T)
40-60% GC content
avoid more than 3 G/Cs in 3’ end
use more G/Cs in 5’ end to make stickier
avoid long runs and repeats over 4 bp
avoid secondary structures (palindromes, inter primer homology)
explain primer dimers
when two primers have homology on the 3’ end a primer dimer forms where the two primers pair up and there is a 3’ OH available for polymerase to bind and extend upon. You end up with lots of amplifications of the primers as your PCR product
what is master mix
add all component of PCR reaction to one tube EXCEPT DNA and primers. saves time and reduces pipetting errors
controls in PCR
positive: contains known amount of target sequence. checks for effectiveness of polymerase and other reagents
negative: contains DNA but does not have target sequence. checks for non-specific priming and amplification
reagent: has everything except DNA. checks for contamination in reagents
how to avoid contamination in PCR
wear gloves
be careful with amplified product
use filter tips
clean hood
make aliquots of reagents so whole supply won’t be contaminated
set up three separate work stations: sample prep (DNA isolation), PCR setup, and analysis on gels
common polymerases
Taq: oldest and most common. no proofreading, low fidelity! Leaves a 3’ A overhang
Pfu, Vent, Pfx: 12X fidelity of Taq, low processivity, slow
Phusion: high processivity, fast, 50-100X fidelity of Taq
Q5: highest fidelity, 100-200X of Taq
how is polymerase fidelity calculated
amplify a gene (LacZ) that turns colonies blue in presence of right substrate, clone product, count white and blue colonies. Sequence to look for mutations
why is cycle 1 the most critical to specificity in PCR?
as temp rises to denaturation temp, polymerase can begin extending primers that may have annealed to non specific sites. Any misprizing mistake in first cycle will be amplified a billion fold by end of reaction.
what is hot start PCR
when a critical component is added or activated only when the reaction has reached 95 C. eliminates possibility of polymerase creating false priming errors as temp ramps up
three methods of hot start PCR
- add critical component for PCR reaction once denaturation temp has been reached
- separate components with wax
- modify Taq DNA polymerase, inactivating it at low temps. can by done by non-covalently bound inhibitor that blocks active site until high temp causes it to dissociation or by covalent mods of the polymerase active site that inhibit activity until heat or pH change causes mods to be hydrolyzed
conditions that may need to be optimized for PCR
primer concentrations
annealing temp
Mg concentration
run PCR in a range of the conditions and see which conditions give best results on gel
what is quantitative PCR
use a DNA binding fluorescent dye or probe to measure fluorescence after each PCR cycle, tells you the amount of DNA present in a sample
what is digital PCR
a way to quantify DNA. run many duplicate reactions but dilute the sample to the point that most reactions will have no template. count number of positive reactions and do some statistics to determine number of molecules you started with
can use microfluidic chip or digital droplet PCR for same thing
what is isothermal amplification
loop mediated isothermal amplification is a newer technique that amplifies DNA like PCR, but with no thermal cycling. uses 4-6 primers and is more difficult to optimize, but has way less instrumentation need and is more useful outside the lab
what is electrophoresis
allows separation of biomolecules based on size. samples are loaded on separation matrix (gel) and an electric field is applied. neg charged biomolecules travel toward the cathode. larger molecules travel slower
standard gels separate fragments from 100 bp to 20,000 bp. pulsed field gels separate super large fragments (10,000,000 bp)
types of gels
polyacrylamide: uses acrylamide monomers (toxic) polymerized to form matrix. pore size defined by concentration of gel, generally 5-15% acrylamide. very high resolution, suitable for 6 bp to 1000 bp fragments
agarose gel: isolated from seaweed, purified agar, percentage agarose depends on fragment size. .5-10 kb 1%, larger 0.8%, smaller 2%. high percent = decrease pose size, harder to migrate.
what affects DNA mobility/migration? besides gel %
conformation of the DNA (supercoiled, nicked circles, linear)
voltage
type of agarose (variable melting temps, etc)
buffer
properties and types of running buffer
gel is saturated with an electrically conductive buffer that establishes and maintains proper pH and provides ions to support conductivity. without it, no migration occurs, if it is too concentration, you may melt the gel
Most common:
Tris Acetate EDTA - low ionic strength and buffering capacity, good for large fragments
Tris Borate EDTA - high ionic strength and buffering capacity, good for small fragments and long runs
Others:
Sodium Borate - fast
Lithium Borate
why does buffering capacity matter
some gels must run for a long period of time. buffer can get polarized at each end of the gel and resist the current. energy is released as heat and gel can melt. TBE has better buffering capacity and won’t overheat in long runs
properties and types of loading buffer
high density to make sample stay in the well. allows a visual estimate of the rate of travel.
Bromophenol blue - migrates like a 300 bp fragment, can leave shadow in UV
Xylene cyanol - migrates with 4000 bp fragment
Orange G - migrates with 50 bp fragments
visualization options for gels
ethidium bromide: works by intercalating in stacked base pairs, elongating DNA helix. fluorescence increases 20X upon DNA binding. stained bands visualized by UV
SYBR Safe: comparable sensitivity as EtBr but less mutagenic. illuminated with blue light (no UV damage)
SYBR Gold: more than 10 fold more sensitive than EtBr, but expensive
methylene blue: non toxic, long protocol, low sensitivity
silver staining: high sensitivity, long protocol, also stains proteins
what is capillary electrophoresis
sample is electrophoresed through a capillary filled with gel. fluorescence is detected electronically in the capillary and converted to an ectropherogram. good for high capacity/throughput and can precisely quantify and size bands
BioAnalyzer (microfluidic chip) functions similarly
