Exam 2 Flashcards
how to identify the fragment of interest in a DNA gel extraction
Place gel on a light box appropriate for the dye used. Avoid exposing DNA to mutagenic radiation for longer than necessary (UV for EtBr).
how to isolate a DNA band from a gel
Physically remove DNA band of interest with a razor blade. minimize the amount of gel around the band that is excised
how to clean up an isolated DNA band via electroelution
recover DNA fragments from a particular region of agarose or polyacrylamide gel. The gel piece is placed in a dialysis bag with buffer. Electrophoresis causes DNA to migrate out of the gel into the dialysis bag buffer. Recover fragments and purify using phenol/chloroform extraction followed by ethanol precipitation
simple, rapid, high yield method
how to clean up isolated DNA fragment via spin column extraction
Dissolution of gel slice at 50 C. DNA adsorption on silica membrane or beads in high salt buffer. Ethanol washes salt and impurities out of column, DNA remains bound. Low salt solution or pure water displaces DNA (elution)
how to clean up an isolated DNA fragment via enzymatic method
Agarase enzyme digests agarose polysaccharide core. Use low melting point agarose to make the gel so you can melt gel at 70 C. Then transfer to 45 C and add enzyme.
DNA can be used directly in many applications without need for further purification
other methods of clean up after gel extraction
E-Gel CloneWell: gel has two rows of wells. insert sample in top well, run until desired fragment is in bottom well, remove from bottom well.
Automated Gel Purification: machine does a very precise extraction. expensive
why is it important to avoid excessive UV exposure when visualizing a gel?
DNA is damaged rapidly by UV radiation. EtBr dye is visualized under a UV light, but exposure for over 30 seconds fatally damages most DNA. SYBRSafe is a good alternative to EtBr because it is visualized under blue light which is not harmful at all.
biological functions of ligases
Lagging strand DNA synthesis
genetic recombination
DNA repair
Ligation reaction components
Buffer: ATP (energy, don't freeze-thaw too much!), DTT (antioxidant), BSA (stabilizer), Mg2+ (enzyme cofactor) Plasmid Insert DNase-free water T4 DNA Ligase (packaged in 50% glycerol)
explain ligation in sticky vs blunt ends
sticky ends: have complementary overhangs which make ligation efficient
blunt ends: no overhangs or complementarity. ligation is inefficient and needs high concentration of ligase and DNA. molecular crowding agents like PEG 8000 can improve intermolecular ligation
intra- vs inter- molecular ligation
intramolecular ligation: within the same molecule (circularization). favored by LOW concentrations of ends. length of molecule is inversely proportional to frequency of ligation
intermolecular ligation: occur between different molecules. dependent on concentration of ends (not plasmid:insert ratio). favored by INCREASE in concentration of ends
explain the results of different concentrations of ends and insert:vector ratios
Very high end concentrations causes concatamers. If insert:vector ratio is high, concatamers of inserts form and vice versa, equal ratio forms plasmid insert concatamer
Very low end concentrations has bad success rate. If high i:v ratio, few colonies have plasmid and none have insert. If i:v low, lots of colonies with plasmid but no insert. If equal, some colonies with plasmid but not insert
Medium end concentration with high i:v has highest product yield when plasmid is dephosphorylated. With low i:v plasmid dimers or recircularizep plasmids form. With equal i:v there’s a 50% chance of correct ligation and highest yield of product
What is Calf Intestinal Alkaline Phosphatase
CIP dephosphorylates 5’ phosphate groups from DNA, RNA, and ribo/deoxyribonucleoside triphosphates. Effect is that DNA cannot self ligate after CIP treatment. This reduces vector background noise and favors clone insert
replaces phosphate with OH group, no phosphodiester linkage can form with self. insert is still phosphorylated so it can form linkage with plasmid (result NICKED circle)
How to calculate ligation reaction concentrations and volumes
i = ratio (insert length bp / plasmid length bp) p
This equation gives an answer of insert = #plasmid. Then you can substitute this equation for i in the equation
i + p = total DNA needed (calculated from reaction volume x total DNA concentration)
to get #p + p = total DNA
solve for p to get ng of plasmid needed. use p to solve for ng of insert needed.
finally, use given plasmid and insert concentrations to calculate volumes in µl
how to use linkers in cloning
use ligase to add linker molecules (containing restriction sites) to your DNA insert. then cut at the restriction sites and insert into a vector with complementary overhangs.
how to use adaptors in cloning
ligate double stranded cDNA with adaptor molecule (short sequence with one blunt end and one sticky end lacking the phosphate) so the blunt ends join together. remove unligated stuff and dimer adaptors. phosphorylate the adaptor-ligated cDNA with a kinase. ligate cDNA to a vector with an overhang complementary to the adaptor
advantage is you do not need to treat the adaptor modified DNA with restriction enzyme
explain genomic DNA libraries
how to make: isolate DNA from cells. digest with REs, nucleases, or mechanically. ligate into vectors and insert into bacteria to amplify. isolate the DNA to form a collection of genomic DNA library.
potentially includes entire genome of the organism and is large and complex. expression is difficult due to introns and possible lack of entire gene. can use any vector type. good to pull out non-expressed sequences (promoters, intons, etc)
explain cDNA libaries
how to make: isolate mRNA and reverse transcribe into cDNA. digest with REs and ligate into vectors. insert into bacteria for amplification and then isolate the DNA for a collection of cDNA library.
includes only the mature mRNA sequences of genes expressed in source tissue. fewer clones, but only the most important sequences. proteins can be readily expressed. clones are small, so use vectors like lambda, plasmid, or cosmid/fosmid.
types of polymerase for PCR
non-proofreading:
Taq. lacks 3 - 5 exonuclease activity. results in a single nucleotide overhang, usually A, which can be cloned into T vectors.
proofreading:
results in blunt ends and must be cloned into a blunt ended vector or needs a single A overhang to ligate into a T vector
advantages and disadvantages of TA cloning
utilizes the unpaired A residue produced by non proofreading DNA polymerase to pair with a linear T vector
Advantage: universal, speed, lack of dependence on REs
Disadvantage: no directionality. must pick and analyze several closes when orientation is needed
how to make a T vector
- digest a vector with a restriction enzyme that generates an unpaired 3’ T residue
- use terminal transferase and ddTTP to add a single T residue to the 3’ termini of vector
- use template independent terminal transferase activity of Taq polymerase to add a T residue to the terminal 3’ end. if only dTTP is present, it will add it instead of an A residue
- purchase a ready made T vector
describe TOPO TA cloning
utilizes topoisomerase of vaccinia virus which relaxes DNA supercoils by transient breaks in one DNA strand. cleavage occurs with high specificity. nicking of the DNA occurs as a result. Because of conserved bond energy, the reaction is readily reversible and the phosphotyrosyl-DNA intermediate can:
catalyze reformation of the original duplex or
create a recombinant DNA by forming a phosphodiester bond
how to clone blunt ends made by proofreading polymerases
TOPO blunt-end cloning. Same process as TOPO TA cloning. The topoisomerase cleaves and then allows for new bond formation. primers are made with no 5’ phosphate so if plasmid is also lacking the 5’ phosphate nothing will happen, make sure to order primer with 5’ phosphate or add one
describe In-Fusion (Clontech) cloning
a recombination-mediated technique for cloning any PCR product into any vector. design primers with an extra 15 bp at the 5’ end that matches the position in the vector where you want to clone the PCR product. Both ends of the PCR product need the matching sequence. With the added vector complement sequence, it’s easy to get the desired construct in a reaction with the In-Fusion enzyme.
Can be used to clone in multiple fragments at the same time
how to fix reading frame problems via PCR
carefully design PCR primers to yield desired RF. plan cloning strategy from the beginning for desired clone. Add or subtract nucleotides from 5’ ends of primers. instead of cutting gene out with REs, amplify by PCR and add RE sites to primers for cloning.
explain ampicillin and resistance
ampicillin is a B-lactam antibiotic that interferes with bacterial cell wall synthesis (B-lactam is part of cell wall)
B-lactamase is an enzyme that breaks the B-lactam ring of the antibiotic and nullifies the antibiotics effects
explain kanamycin
interact with 30S subunit of bacterial ribosomes and induces high levels of mistranslation, indirectly inhibiting translocation during protein synthesis
define transformation
the genetic alteration of a cell (bacteria) caused by the uptake of foreign DNA
what is the barrier that must be overcome for transformation occur
the hydrophobic interior of the phospholipid bilayer of cell membranes prevent polar molecules (DNA) from passing through the membrane. We need a way to get our foreign DNA inside the cell (heat shock/chemical or electroporation)
how to make cells chemically competent
- wash in NaCl - remove ions and contaminants
- CaCl2 added - makes membranes more permeable
- Glycerol added - allows cells to be frozen, preserved in cryostatic state
can also buy competent cells
Heat shock procedure
Keep cells cold! Don’t mix vigorously (even pipetting up and down too much)!
Chill cells on ice (DNA binds to cells)
Heat to 42 C to make membrane fluid, porous, allowing DNA into cell
Chill on ice again
Add SOC medium with no antibiotic. grow 30-60 minutes. gives cells time to grow and produce resistance proteins
Plate on selective media plate
electroporation procedure
Close 1st switch: capacitor charges up and stores a high voltage
Close 2nd switch: voltage is discharged through cell suspension liquid
A typical pulse is 10,000 to 100,000 V/cm lasting a few microseconds to a millisecond
Temporary aqueous pores are formed by the disturbance of the phospholipid bilayer caused by the electric shock and charged DNA molecules are driven across the membrane by increase in electrical potential of the cell membrane
Review the lac operon transcription mech
LacI gene encodes a repressor. If no lactose is present, the repressor will bind the lacZ operator and block transcription. If lactose is present, allolactose will bind the repressor and stop it from blocking the operator. Transcription occurs and B-Galactosidase, permease, and transacetylase are made.
explain alpha complementation in the lac operon
Two fragments of B galactosidase are synthesized:
1. host strain makes inactive C terminal fragment called Omega fragment
2. pUC plasmid makes inactive N terminal fragment called Alpha fragment (This is because pUC plasmids have a size limit of what they can handle, so only part of the lacZ gene fits)
When both fragments are present, B galactosidase activity is observed.
explain blue white screening
B galactosidase produced by the lac operon cleaves X-gal to galactose and an insoluble blue product (blue colonies). If the MCS is inside the lac operon and DNA is inserted, the lacZ gene is disrupted and B galactosidase is not produced, X-gal is not cleaved and colonies are white
what are satellite colonies
colonies that contain no plasmid but grow on ampicillin plates because of secretion of B lactase from plasmid colonies. occurs when you overgrow your colonies (16 hours max)
review the steps in alkaline lysis (explain what ingredients are for in each step!)
- cell growth and harvesting: pick colony and grow in liquid culture, incubate overnight (shaking), pellet cells, and decant medium to dry
- Resusupension: add solution with glucose (osmotic pressure), Tris (pH), and EDTA (destabilize membrane, deactivate DNAses)
- Lysis: add solution with SDS (solubize membrane, denature proteins) and NaOH (break cell wall, denature dsDNA to ssDNA)
- Neutralization: add solution with acetic acid (neutral pH, renature DNA) and potassium acetate (precipitates SDS, debris, and chromosomal DNA)
- Cleaning and concentration: phenol chloroform extraction to remove debris and ethanol to precipitate. resuspend in TE buffer
why is it important to remove all traces of medium when performing prepping for alkaline lysis?
cell wall components remaining in supernatant may inhibit restriction enzyme action on DNA downstream, so remove as much medium as possible
Why are alkaline conditions important in lysis?
alkaline conditions denature DNA. The neutralization step in lysis adds alkaline conditions because then genomic DNA can’t renature, but plasmids CAN because they never fully separate
describe phenol:chloroform extraction
Good general purpose method to clean up DNA/RNA
1. Add 1 volume phenol:chloroform:isoamyl alcohol and vortex
2. centrifuge
3. remove aqueous layer
4. ethanol precipitate
DNA is in the top layer. Proteins will be denatured by the phenol and will be in the organic phase (bottom layer) or white interface
describe alcohol precipitation
DNA/RNA will precipitate in ethanol or isopropyl alcohol if salt is high. good general purpose method to clean or concentrate DNA/RNA
- add 1/10 volume salt
- add 2 - 2.5 volumes EtOH or 1 volume IPA
- vortex and incubate 5 mins, cold helps
- centrifuge 5-10 minutes or longer
- remove supernatant
- wash with 70% ethanol and centrifuge 5 mins
- remove supernatant
- dry until no EtOH is visible and resuspend
describe column based miniprep
resuspend and lyse according to normal protocol. Neutralize with chaotropic salt (guanidinium chloride) instead of potassium acetate, DNA is bound to silica column, wash and elute.
Columns are usually spun in a centrifuge, but many can be used on a vacuum manifold instead. Can also use anion exchange instead of silica (elute in high salt buffer) or magnetic beads coated in DNA binding material
describe the Maxam-Gilbert Chemical Cleavage Method of sequencing
First widely adopted method, but rarely used now because it is not easily scaled and rather tedious. DNA is labeled and then chemically cleaved in a sequence dependent manner (such as cleaves at every A base) then you can run the fragments on a gel and get sequence from the fragment lengths
describe the Sanger Dideoxy method for sequencing
popular, adaptable, and scalable.
- Primer anneals to ssDNA
- extend primer with DNA polymerase in presence of all four dNTPs, with a limited amount of a ddNTP
- DNA polymerase incorporates ddNTP in a template dependent manner, works best if DNA polymerase lacks proofreading. Reaction stops once ddNTP is added
reaction components in Sanger sequencing
- DNA polymerase: processive, no 3 - 5 exonuclease activity
- Template DNA: single stranded DNA from recombinant bacteriophage or denatured dsDNA or non-denatured dsDNA (cycle sequencing)
- Primer: usually designed and purchased, sequence of the oligo designed to begin at a particular location. 15-30 bp length
- dNTPs
- ddNTPs
What is cycle sequencing? Whats the advantage
Cycle sequencing: synthesis reaction (denaturation, annealing, extension) is repeated many times. don’t need lots of DNA and no denaturation prep necessary, but DNA pol may incorporate ddNTPs poorly
Like PCR but with one primer and not exponential (linear amplification e.g. 30 cycles is 30X synthesis)
Big advantage is that you don’t need to get ssDNA before sequencing.
how to visualize DNA fragments in Sanger sequencing?
Radioactivity: radiolabel primers and radiolabel dNTPs. Four reactions needed, one for each nucleotide (same signal for all four nucleotides)
Fluorescence: ddNTPs made to contain fluors. Each ddNTP fluoresces at a different wavelength, so all four reactions can be run in the same tube
how to analyze radiolabeled Sanger sequencing products
analyze with polyacrylamide gel electrophoresis. Must have good resolution of fragments differing be a single dNTP. Start reading sequence from the BOTTOM of the gel (shortest fragment to longest fragment)
Slab gels: ultra thin gel between two glass plats. Not common today
Capillary gels: require only a tiny amount of sample to be loaded, run fast, best for high throughput sequencing.
how to analyze fluorescent labeled Sanger sequencing product
Two options: label the primer or the ddNTPs
- Primer labelled with a different color for each ddNTP reaction. four reactions, each with different ddNTP label, all loaded into the same lane
- Each ddNTP labelled with a different dye allows for one reaction and one lane
how to read a chromatogram and why is separation worse with longer fragments?
slide 23 (look on canvas) for reading chromatogram large fragments are harder for the machine to distinguish because the sizes are very similar. A 700 bp fragment is super close in size to a 701 bp fragment, where a 10 bp fragment is easily different from an 11 bp fragment. So at about 700 bp, separation is not as good. At about 1000 it is very bad.
what is DNA foot printing or DNAse protection assay
identifies binding sites of DNA binding proteins. Labeled DNA fragment is partially digested by DNAse in presence of DNA binding protein. DNA is protected from cleavage in the area where protein is bound. Run on a gel: DNA digested with protein bound, DNA digesting without protein, and DNA sequencing reactions
Result: you can see a gap in fragment bands where the protein was bound. compare to sequence reaction for protein binding sequence
what is primer extension
identifies 5’ end of transcribed RNA. Labeled primer is bound to mRNA molecule and then reverse transcribed to make a single length product, corresponding to the distance from the 5’ end of the transcript to the primer. Run on gel next to sequencing reactions with same primer.
Band lines up with the sequence band that is the 1st base in the 5’ end of the mRNA transcript
brief history of cell culture
Wilhelm Roux: maintained a portion of chicken embryo in vitro
Ross Harrison: maintained embryonic frog neural crest fragments in a drop of coagulated frog lymph for several weeks. observed in vitro growth of nerve fibers
Rous & Jones: used trypsin to make unicellular suspensions
Carrel & Baker: T flasks and techniques for scale up of culture
Fischer: developed chemically defined medium (CMRL 1066)
John Enders: growth of virus in culture. Polio virus HPV
Harry Rage: developed Minimal Essential Medium (MEM) commonly used today
structural things to note about animal cells in culture
10 to 100 microns in size and spherical shape in suspension. No cell wall. Plasma membrane is thin and fragile and shear sensitive. Surface is negatively charged and so is grown on positively charged surfaces
What is transfection and why do it?
Introduction of foreign nucleic acids into mammalian cells in order to produce genetically altered cells.
Used to study:
Gene expression
Protein function
Cell function
Mark cells to follow migration, death, morphology, etc
Gene Therapy
transient vs stable transfection
transient: DNA introduced into cell line generates a temporary but high level of gene expression. DNA is not integrated into genome. good method when large sample numbers need to be analyzed in short time
stable: establishes clonal cell lines where DNA is integrated ingot he cells genomic DNA. Target protein is synthesized in moderate amounts. Much lower efficiency, isolation is facilitated by selectable marker
Controls that should be included in all transfections
Individual reagent controls
Plasmid DNA preparations
Toxicity of the gene or construct
Transient expression controls
Negative: cells are transfected with carrier DNA and/or the buffer used to dilute plasmid or gene. Empty vectors in cells should result in cells not detaching from dish or becoming rounded and glassy. CHECKS FOR CONTAMINATION
Positive: cells are transfected with a plasmid encoding an assay product, like fluorescent protein. Need to be able to detect products and proteins. CHECK THAT INSERT IS GETTING INSIDE CELLS.
Stable expression controls
Negative: cells transfected with an inert nucleic acid with no selectable marker. No colonies should be visible. CHECK THAT SELECTION PROCESS WORKS
Positive: cells transfected with empty plasmid encoding selectable marker. number of colonies after 2-3 week culture is measure of transfection efficiency. Large discrepancy between this control and the test plate could indicate toxic gene product.
describe optimization considerations in transfections
Antibiotic concentration: generate kill curve to see best amount.
Cell type: diff cell lines vary in ability to take up and express DNA. Some conditions may work for one line but not another. Assess cell viability when optimizing cell type
How to assess cell viability
Cytotoxic assay: number of dead cells
Viability assay: number of live cells
Total number of cells
Mechanism of cell death (apoptosis vs necrosis)
Indicators of cellular integrity: DNA content, enzyme activity, ATP amount, membrane integrity metabolic activity
three categories of transfection methods
Biochemical methods: includes Calcium phosphate mediated, DEAE dextran, and cationic/lipsome
Physical methods: includes direct microinjection, electroporation, biolistic particle delivery, optical transfection, and hydrodynamic
Virus mediated
what affects your choice of transfection method?
Whether gene expression or protein production is the desired end result
Cell type to be used
Ability of the cell to survive the stress of transfection
Type of assay to be used for screening
Culture Conditions
Nucleic acid to be transfected (DNA, RNA, siRNA, oligonucleotides)
Efficiency required of the system
Throughput needs
Describe biochemical transfection methods
Calcium phosphate or DEAE dextran was historically popular. Lipid agents are commonly chosen now because:
high efficiency
ability to mediate transfection of all nucleic acids in a wide range of cell types
ease of use, reproducibility, and low toxicity
can mediate transfection of suspension cells
+ chemicals form complexes with - nucleic acids, attracted to - membrane, passes through membrane to nucleus
pros and cons of biochemical methods of transfection
cons: low transfection efficiency, especially in vivo. efficiency depends on nucleic acid/chemical ratio, pH, and cell membrane conditions
pros: low cytotoxicity, no mutagenesis, no extra-carrying DNA, no size limitation
Describe physical methods of transfection
consider with cell lines that are resistant to transfection by other means. often much harder on the cell
Direct microinjection: put nucleic acid directly into cell. demands skill, often causes cell death, and is very labor intensive
Biolistic particle delivery: gold particles conjugate with nucleic acids and are shot into cells at high velocity. straightforward and reliable, but expensive and cause damage
Electroporation: short electrical pulse disturbs membranes and make holes for nucleic acids to pass through. easy and rapid. can transfect large numbers of cells fast. highly efficient
Describe virus mediated methods of transfection
TRANSDUCTION
high efficiency and easy to achieve trans gene expression in vivo because of integration of viral genome into host genome. effective on dissociated cells/slices
potential hazard to lab personnel, immunogenicity and cytotoxicity, insertional mutagenesis (random integration can alter host genes), small size of virus limits size of foreign gene
general goals of genomic DNA purification
Removal of proteins, removal of RNA, isolation of a specific type of DNA, high final concentration, high molecular weight
more or less important depending on downstream application
types of DNA and where they are found
Genomic: chromosomal Organellar: eukaryotes only (mitochondria) Plasmid: extra chromosomal Phage/viral cDNA: made from mRNA
uses of genomic DNA
southern blotting
cloning
library construction
template for PCR (amplify region for research, genotyping, DNA fingerprinting)
general features of ALL genomic DNA purification methods
Tissue disruption
Cell lysis
Protein denaturation/removal
DNA concentration
describe tissue disruption
Can be difficult, so if only genomic DNA is needed choose a tissue that is easy to obtain and disrupt. Blood and cultured cells only require lysis, other tissues may need mechanical or enzymatic disruption.
Mechanical: blenders, shakers, pestles, etc break tissue to small pieces
Enzymatic: good for connective tissue. Proteinase K or Collagenase digest tissue
notes about plant cell lysis
cell wall makes disruption more difficult. use mechanical disruption and boil in a solution with a Ca2+ chelator and detergent
common cell lysis buffer components
Guanidinium thiocyanate: protein denaturant
Guanidinium chloride: protein denaturant
Detergents like SDS or Triton X-100: solubizes membranes and denatures protein (only SDS)
salt and pH buffer
describe salting-out as a DNA extraction purification
proteins and other contaminants are precipitated with high concentrations of salt (potassium acetate or sodium acetate). highly variable purity and yield, but simple and fast
describe organic extraction method of DNA purification
Phenol denatures and precipitates proteins, mix gently to avoid shearing DNA. remove aqueous top layer (contains DNA/RNA) and extract again with chloroform (removes phenol).
describe lysis and sequestration method of DNA purification
Goal is to homogenize tissue in lysis buffer designed for downstream uses. Very simple methods, quick and inexpensive, but low purity. DNA may not be stable for long term storage and buffer components may not be compatible with downstream applications, requiring further purification
describe adsorption method of DNA purification
Nucleic acids adsorb to silica or other resins in the presence of chaotropic agents or salts. Formats can include resins beads, centrifuge columns, and magnetic beads. DNA is eluted in a low salt buffer or water (removes chaotropic salt which allows DNA to fall off silica)
describe anion exchange as DNA purification method
In a low salt buffer, DNA binds to the anion exchange column. Wash with a low salt buffer to remove impurities. Elute DNA off column with high salt buffer (replaces DNA on resin) and precipitate DNA with alcohol
describe density gradient centrifugation as a DNA purification method.
DNA is mixed with EtBr and ultracentrifuged for hours in a gradient medium. DNA migrates to a defined location based on density. EtBr used to visualize. Desired band is removed
Most dense (lowest) to least dense (highest) in column: RNA, ssDNA, dsDNA, protein
how is DNA precipitated? (ethanol)
DNA does not dissolve in alcohol. Salt allows DNA molecules to come together to aid precipitation in alcohol. Alcohol causes DNA to clump and precipitate as long, fluffy, web like strands. oxygen bubbles aggregate and lift DNA out of solution into organic phase
how to extract chromosomes
block cells at metaphase and lyse gently
procedure for high molecular weight genomic DNA purification
vortexing and pipetting break DNA into ~40,000 bp fragments, so must be very gentle if you want larger fragments
- Cell lysis: SDS and Proteinase K
- Phenol extraction with gentle rocking for several hours
- Ethanol precipitation
- RNAse followed by proteinase K
- Repeat phenol extraction and EtOH precipitation
What is one extra substance that should be added when extracting plant DNA?
CTAB (a cationic detergent) to remove carbohydrates that interfere with downstream processes
