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Biol 208 Midterm 2 (module 6-12)

18 Pages

Course Code
BIOL 309
Kyra Jones

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Bio 208 Midterm 2 NotesModule 6 Cloning Vectors vector nucleic acid molecule used to transport and replicate sequences of interest into a host organismProperties of Plasmid Vectors1small25kb the larger the insert the small the vector2origin of replicationplasmid must be able to replicate in the host cell determines the copy number high copy number means slow growth a low copy number means fast growth and greater stability 3unique cloning siteMCS have multiple recognition sites4selectable markersa gene on the plasmid will only allow successful transformants to grow in a certain environment ex Antibiotic resistance5complete sequence of vector is knownrestriction enzyme recognition sites ability to design primers for amplification of unknown sequences6insertional activation sitesalpha complementation lac Z is a partial Bgalactosidase gene from the lac operon the other parts of the enzyme are supplied by a specific host strainlacZ expression is induced by growth on IPTG isopropylbetaDthiogalactopyranoside and turns blue in the presence of Xgalthus white colonies indicates acomplementation is not occurring if a successful insert was cloned into the MCS within Lac Z7promoters flanking the multicloning regionadditional promoters flanking the MCS allow the insert to be transcribed under specific conditionsriboprobesenable singlestranded probes to be generated in vitro sense or antisenseoverexpressioncontrolled protein expression small expression to allow cell growth then once cells are plentiful the protein is greatly expressed 8reporter genes or epitope tagshis or myc tags have green fluorescent protein or epitope tags used for protein localization and affinity purificationSubcloning1digest parental vector to remove insert2run on agarose gel3remove gene of interest by cutting from gel and purifying with an affinity column4PCR to amplify5ligate insert with cut destination vector with T4 DNA ligase6transform7plate on selective media or screen on Xgal8miniprep digestTransformation MethodsCaCl2 Treatment Followed by Heat ShockCaCl2 affects the cell wall allowing DNA binding heat shock stimulates DNA uptakeLB is added after heat shock to allow time for the marker to be expressed1grow cells to log phase2collect by centrifugation wash and resuspend in cold CaCl23add naked DNA4heat shock at 42C for 1 min5add LB and let grow for 1 hr at 37C6plate on selective mediaElectroporation wash with ice cold water cold prevents cell growth to remove electrolyteshigh voltage opens pores allowing DNA entrylonger preparation but is faster and has a higher success rate than CaCl2 treatment1grow cells to log phase2collect by centrifugation wash several times with water3resuspend at high conc in 10 glycerol and freeze in small aliquots using liquid Nitrogen or an ethanol bath on dry ice 4mix frozen cells with DNA in a cuvette5zap with high voltage briefly msec6add LB and let grow for 1 hour at 37C7plate on selective mediato test competency a known amount of supercoiled plasmid is added ex add 5ng of pUC18 to 01mL of CaCl2treated Ecoli cells with 1mL LB broth and plate 50uL and 5uL of transformants on selective media137 colonies found on 50uL and 18 on 5uL plate01mL cells1mL broth11mL total50uL005mL11mL005mL22 plates22 plates x 137 colonies3x103 transformants If 5 ng of DNA produces that many colonies how much would 1ug DNA produce3x1030005ugx1ug x6x105 transformants improving transformation efficiencymodify salt solution add salts like Mg to CaCl2 or use specialized host strains with endonucleases that prevent digestion of the plasmid or recombination genes that reduce recombination of the plasmid and improving plasmid stabilityBacteriophage LambdaLytic Cycle1bind to protein receptor on cell wall2injects linear DNA into cell genome circularizes prevent degradation via cohesive ends3initial bidirectional theta replication4subsequent rolling circle replication generates multiple copies of the genome including cos sites called concatemers 5adjacent cos sites recognized by viral packaging machinery leads to insertion of the linear genome into capsidsLysogenic Cycle1binds to protein receptor on cell wall2injects linear DNA into cell genome circularizes via cohesive ends3if there is accumulation of the cI repressor the genome inserts by specific
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