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60 Cards in this Set
- Front
- Back
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Plasmid vectors?
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>1 selectable marker
-resistance to antibiotic or intro of gene encoding enzyme Origin of replication -allows replication of extra chromosomal DNA Unique restriction enzyme cutting site (multiple cloning site) -insertion of DNA into plasmid |
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Why pBK-CMV?
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17 unique restriction enzyme cutting sites
Neomycin resistance gene allows selection in euks and proks -lac promoter drives prok & cytomegalovirus immediate early promoter drives euk |
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unit
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amount of enzyme needed to completely digest 1µl DNA in 1 hour at 37C
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why restriction enzymes stored in 50% glycerol
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prevents freezing at -20C & most stable at cold temperatures
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buffers contain usually
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Tris, Mg2+, proper salt concentration
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why not polyacrylamide and agarose
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polyacrylamide good for both nucleic acids & proteins and good resolution, but difficult to pour & neurotoxin
kelp polysaccharide and can be melted in hot buffer |
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making the gel
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0.5g 1% agarose (.4-8kb) & 50ml TAE buffer
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load dye's purposes
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glycerol increases sample's density ensuring DNA drops into well
bromophenol blue & xylene cyanol add color to make loading easier dyes move toward anode at predictable rate(b 2.2x faster than x) |
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ethidium bromide for what
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method for straining DNA
mutagen by intercalating between stacked bases UV (260-360mm wavelength) lamp emits light & emits in red orange wavelength |
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calculate titer
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(number of plaques) / (total volume) X Dilution Factor
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plates needed to be screened to ensure P% genome represented
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N=ln(1-P)/ln(1-I/G)
N/(pfu/plate)=plates |
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what's needed to test endonuclease/methylase system
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lambda DNA, restriction enzymes, methylase, 2X SAM buffer (methyl donor), water, buffer (optimal salt concentration) specific for restriction enzyme
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needed for restriction digest
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DNA, 10X buffer (diluted to 1X buffer), water, restriction enzyme
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ways DNA can be introduced into bacteria
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Transformation
-cell's uptake of purified DNA Transduction -intro by bacteriophage Conjugation -transfer of plasmid DNA cell-cell contact Electroporation |
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Types of competence
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Transformation
Artifcial Natural |
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difference between cDNA & genomic library
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genomic represents all DNA but cDNA made from RNA so only genes expressed proteins
cDNA no include introns & can have tissue specific library cDNA library construction little bit more complicated |
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partial digest?
four ways to do it? why do it? |
unequal rate of cutting restriction sites
Limit digestion time Vary buffer Limit amount of restriction enzyme Vary incubation temperature obtain different sizes of overlapping DNA that encompasses gene of interest & obtain DNA suitable size for cloning |
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ZAP Express features
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Left & right arms contain necessary info enabling phage to infect bacteria, allows replication of phage & proper packaging of new phage & replicate other bacteria once lysed inf first
Middle region of lambda phage DNA necessary for lysogenic phage removed replaced with plasmid pBK-CMV |
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components of stop solution
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EDTA
10X loading dye |
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titer's importance
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reveal if library contains enough viable phage to include all regions
in order to screen library certain phage density needs to be plated |
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plaque purification
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positive plaques identified via probe hybridization with gene of interest picked & replated at higher dilutions & rescreened to yield positive single plaques
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why plate serial dilutions for titer
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make sure you get plates you can count & confirm titer calculated from one dilution with other dilutions
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why important to add 0.2% maltose & 10mM MgSO4 before adding E. Coli phage
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maltose for increase rate of infection by phage on bacterial cells & induces maltose operon in bacteria & leads to increase of maltose receptors (sites of infection for phage)
MgSO4 stabilizes interaction between phage & maltose receptor |
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plaque lifts done on top agarose, not top agar, why
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top agar can tear off plate easily
top agarose adheres to bottom agar & more extensively purified & more solid support |
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importance of alkali denaturation step & UV crosslinking step for plaque lifts
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alkali denaturation step to disrupt phage releasing DNA & denature dsDNA into ssDNA adhering to filters
UV crosslinking to anchor DNA to filter in ss state, UV irradiation forms covalent links between thymidine residues in DNA & positively charged groups on filter |
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stringency
when use low/high how increase |
level of specificity chosen for hybridization
High allows probe to bind to exact target & used if sequence is exact complementary to target Low allows more basepair mismatches & used if unknown if matches increase by decreasing cat ions or salt concentration, use denaturants, increase T close to Tm |
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added to prevent non-specific binding of probe to filters
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pre hybridization solution blocks empty areas in filter
lots of sonicated salmon sperm more likely binds to any non-specific areas on filter & prevents probe from being pulled out of solution |
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SSC's purpose
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NaCl, Na citrate
last wash of filters to wash away excess base & acid from denaturation & neutralization washes and keep filters moist last washes during probe hybridization, lower cat ion concentration allows high stringency so more repulsion between negative probe & negative target sequence on filter and allows only perfectly complementary matches |
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Tween's purpose in visualization of hybridized probe
blocking buffer's purpose wash buffer's purpose |
disrupts any non-specific binding of antibody to filters
occupy all non-specific protein binding sites on filters removes unbound antibody so no false positive signal |
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features of Ex-Assist phage necessary for co-infection with ZAP Express into XLI blue M.RF' strain
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replication deficient so easier to identify plaques with ZAP in XLOLR strain (only Ex-Assist not Zap can infect XLOLR & want Zap DNA in Ex-Assist phages)
Ex-Assist express gene for protein cuts at initiator & terminator sequences flanking recombinant DNA has to be excised out from rest of phage DNA |
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in Qiagen plasmid prep, importance of Rnase
N3 EB |
chew up cellular RNA from bacterial since RNA can interfere with further manipulations of plasmid & RNA will show up as spurious band
N3 neutralizes NaOH from P2 (lysis buffer) with acetate & precipitates proteins with high salt after spun, pellet contains cellular proteins & lipids while supernatant contains genomic DNA & recombinant plasmid DNA Elution buffer solubilizes plasmid DNA while genomic DNA is not solubilized & remains bound to column |
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importance of excise out pBK-CMV as well as T cruzi from Zap
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contains kanamycin resistance gene which is important when XLOLR plated on LB+kanamycin
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important when cloning insert into vector to express encoded protein
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proper orientation so promoter is before beginning of gene sequence
correct reading frame |
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why do restriction analysis
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size of insert matches expected for gene of interest
what restriction enzymes cut out insert intact from recombinant plasmid general info about gene of interest |
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difference between variable length strands & primer defined length strands in PCR
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variable length occurs when newly synthesized strand extended beyond primer binding sites; made during initial cycles & increase at arithmetic rate
primer defined occur when newly synthesized strand defined by two primers, primers extended to binding site of second primer; made during subsequent cycles & exponentially increase |
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Taq pol used instead of Klentaq-1 or Deep Vent pol
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Taq thermo stable but others don't & have 3'-5' Exonuclease activity
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primers in PCR should have
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18-30 nucleotides long
50-60% G-C rich same melting temperature |
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purpose of adding Chelex beads during isolation of cheek cell DNA
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binds & chelates heavy metal ions which can inhibit PCR by acting as catalysts in DNA breakdown at high T
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necessary for PCR
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template DNA, 2 primers, Taq DNA pol, buffer, dNTPs
PCR mix (Taq, buffer, dNTPs, MgCl2) |
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competence induced by
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adding CaCl before adding DNA
calcium ions complex with phosphates in cell membrane & destabilize it causing pore formation then complexes with DNA & cling to surface of cell and heat shocking allows DNA into cell |
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efficiency of transformation
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total number of cells/DNA spread on plate
DNA spread= DNA in original X concentration of DNA X dilution |
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Construct DNA library
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Fragmentation
Clone into lambda vector Recombine into complete phage Calculate library titer Plate 50000 pfu Hybridize Visualize Screen Clone into pBK-CMV plasmid |
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Types of vectors
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Plasmids
Bacteriophages Cosmids Phagemids Artificial chromosomes |
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Fragmentation & insert into ZAP
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Partial digest with Sau3A
Fragments purified by density gradient centrifugation Phage arms treated with alkaline phosphatase to remove 5' phosphate to prevent self-ligation & reduce background of non-recombinant vectors Insert into ZAP with BamH1 |
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Why filters washed with 1.5M NaCl/0.5M NaOH & 1.5M NaCl/0.5M Tris-HCl pH 8.0
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Disrupts phage & denatures DNA adhering to filter
Neutralize |
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Types of oligonucleotide probes
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Single oligo of defined sequence
Pools of short oligos with high degeneracy Pools of longer oligos with lesser degeneracy |
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Melting temperature
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1/2 DNA ss
2(A+T)+4(G+C)-10 |
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Novel surfactant
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Prevents probe from binding to filter but doesn't interfere with interaction with target sequence
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Labeling for long probes
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Polymerases to incorporate nucleotides modified to contain radio or nonradioactive label
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Labeling for short probes
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End-labeled with 32P by kinase
Nonradioactive labels inserted during synthesis |
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Cons of radioactive labeling
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Half-life 14 days
Licensing Safety & disposal |
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Colorimetric detection system
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Probe modified with digoxigenin so highly specific
Anti-DIG-AP binds to label AP catalyses removal of phosphate from 5-bromo-4-chloro-3 inoldyl phosphate which dimerizes to from blue precipitate & hydride reduces nitroblue tetrazolium to form purple |
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when phage is mixed with ExAssist what processes are occurring
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ZAP Express infection causing cell to replicate ZAP info
ExAssist helper phage binds to initiator & nicks one of DNA then polymerase replicates other strand & hits terminator & nicks & falls off-> ZAP & ssDNA with PBK-CMV & T.cruzi insert pBK packaged into ExAssist coats |
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supernatant of ZAP, pBK-CMV, T.Cruzi contains
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ZAP with its own DNA
ExAssist phage without plasmid DNA ExAssist with plamid DNA |
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P1
PB PE EB |
resuspension buffer & rnase
binding buffer wash buffer elution buffer |
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how to eliminate wrong orientation
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direction cloning with two different restriction enzymes
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who PCR
what is it |
Kary Mullis of Cetus Corporation
in vitro for enzymatic synthesis of specific DNA sequence template DNA denatured at 95C for 1-3 min then cooled to allow two oligo primers to anneal to target & provide 3'OH required for DNA synthesis primers extended with DNA pol |
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PCR pol
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thermophilic bacterium Thermus aquaticus
95C extension at 75C |
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VNTR
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variable number of tandem repeats
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calculate tandem repeats
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(observed band size-142)/16
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