9052_dna Isolation Report Complete

Samuel'Hourigan' 1FRIX' SID:'430330362'

DNA$ISOLATION$REPORT$ 1''

DNA$ISOLATION$REPORT$

Report$Item$1:$Summary$Table$$

FIGURE$1.$Summary'table;'detailing'yield'and'quality'of'DNA'Isolation'procedure'on'E.'coli'culture'as'detailed'in'figure'2'legend.$

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'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''1'Expected'yield'was'2.5mg.''The'E.'coli'genome'contains'4.610!'base'pairs,'with'an'average'molar'mass'of'a'base'pair'being'660g.'Firstly'calculate'the'molecular'mass'of'a'single'base'pair;' (!!")(!.!""!"!") = 1.09598140210!!"!'From'this'the'molecular'mass'of'a'single'DNA'molecule'may'be'attained; 1.09598140210!!" 4.610! = 5!"!!"#!!"#$%$!!"!!"#'Then'to'calculate'the'expected'yield'the'number'of'cells'in'the'starting'0.5g'of'wet'cells'must'be'attained.''E.'coli'culture'contains'310!"'cells'per'litre'and'yield'~3g'of'wet'weight'of'cells.'Therefore'!.!! 310!" = 510!!'cells'in'0.5g'of'wet'cells.'Finally'the'yield'is'calculated'by'multiplying'the'number'of'cells'in'0.5g'of'wet'cells'by'the'molecular'mass'of'a'single'DNA'molecule.' 510!! 5!" = 2.5!"''

Final'volume'of'DNA'preparation'(mL)'

1'!!"#' 0.692'!!"#' 0.335'!!"#:!!"#' 2.1'Dilution'factor'used'for'UV'spectrum'

20'(150'L'in'3mL)'

Diluted'[DNA]'(g/mL)' 34.6'Original'[DNA]'(mg/mL)' 0.692'Yield'(mg)1' 0.692'Yield'(mg/g'wet'weight'cells)' 1.384'

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Report$item$2:$DNA$Spectrum$with$Legend$

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Figure$2.'A'culture'of'E.'coli'(~0.1'g'wet'weight/mL)'suspended'on'0.1M'NaCl,'10'mM'EDTA,'pH'8'was'incubated'with'2%(w/v)'sodium'dodecyl'sulphate'(SDS)'at'65C'for''15'minutes.'Following'the'incubation,'sodium'perchlorate'was'added'to'1M'and'the'solution'was'extracted'with'an'equal'volume'of'chloroform:'isoamyl'alcohol'(24:1).'After'shaking'for'20'minutes'at'room'temperature'with'a'wristbarm'shaker'the'upper'aqueous'layer'was'removed.'Two'volumes'of'ethanol'were'layered'carefully'onto'this'aqueous'solution'and'the'DNA'was'collected'from'the'interface.'The'precipitate'was'washed'briefly'in'70%'(v/v)'ethanol'then'dissolved'in'TE'(10'mM'Tris,'1mM'EDTA,'pH'8;'0.2mL'TE'per'mL'initial'E.coli'culture).'The'purity'and'yield'of'the'nucleic'acid'preparation'was'assessed'by'UV'spectrophotometry'and'by'gel'electrophoresis,'with'and'without'RNase'treatment.'

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0.000'0.100'0.200'0.300'0.400'0.500'0.600'0.700'0.800'

230' 250' 270' 290' 310' 330'

Absorban

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Wavelength$(nm)$

DNA$Absorbance$Spectrum$A260:A280=$2.1$

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Report$item$3:$New$Protocol$

Figure$3.$Original'and'New'protocol'comparison.'New'protocol'altered'from'the'original'protocol'based'upon'the'change'from'5mL'(~0.5g'wet'weight'packed'cells)'starting'volume'and'25%(w/v)'stock'concentration'of'SDS,'to'25mL'(~2.5g'wet'weight'packed'cells)'starting'volume'and'20%(w/v)'stock'concentration'of'SDS.'(Items'changed'for'the'new'protocol'are'highlighted)$

Original$protocol$(5ml$starting$volume,$25%$SDS)$ New$protocol$(25mL$starting$volume,$20%$SDS)$1. Cell$Lysis$

$ Each'pair'is'provided'with'5mL'of'E.'coli'suspension'(dissolved'in'0.1M'NaCl,'

10mM'EDTA,'pH'8)'containing'~0.5g'wet'weight'of'packed'cells.''

Add'0.4mL'25%(w/v)'SDS'(sodium'dodecyl'sulphate)'to'the'suspension.''

' Incubate'the'mixture'at'65'for'15'minutes.'At'the'end'of'the'incubation'the'

solution'should'be'very'viscous'as'you'have'lysed'the'cells'and,'hopefully,'released'the'cell'contents'into'the'solution,'including'the'DNA.'The'SDS'(detergent)'will'also'help'dissociate'the'proteins'from'the'DNA.$

1. Cell$Lysis$$ Each'pair'is'provided'with'25mL'of'E.'coli'suspension'(dissolved'in'0.1M'

NaCl,'10mM'EDTA,'pH'8)'containing'~2.5g'wet'weight'of'packed'cells''

Add'2.8mL'20%(w/v)'SDS'(sodium'dodecyl'sulphate)'to'the'suspension.''

' Incubate'the'mixture'at'65'for'15'minutes.'At'the'end'of'the'incubation'the'

solution'should'be'very'viscous'as'you'have'lysed'the'cells'and,'hopefully,'released'the'cell'contents'into'the'solution,'including'the'DNA.'The'SDS'(detergent)'will'also'help'dissociate'the'proteins'from'the'DNA.$

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2.$Removal$of$protein$ Add'1.3mL'5M'Na'perchlorate'(!"#$!!)'and'mix'well'by'inversion'(try'not'

to'create'bubbles).'The'high'ionic'strength'disrupts'the'DNA'protein'interactions'and'helps'to'remove'the'proteins'from'the'nucleic'acid.''

Add'an'equal'volume'of'chloroform:isoamyl'alcohol'solution'(24:1)'and'firmly'screw'down'the'lid.'Chloroform'is'nasty'stuff'(carcinogenic'etc.)'and'is'stored'in'the'fumehood.'A'demonstrator'will'do'this'addition'for'you'in'the'fumehood.'Only'take'the'tube'out'once'the'lid'is'tightly'screwed'on.'

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Invert'the'tube'by'hand'continuously'for'5Z10'minutes''wear'gloves'and'ensure'the'lid'remains'tightly'sealed'onto'the'tube!'This'mixing'step'is'more'vigorous'than'using'a'mechanical'shaker.''

At'the'end'of'the'mixing'step,'allow'the'tube'contents'to'settle'(~5Z10'minutes).'Remove'the'bottom'chloroform'layer'(chloroform'is'denser'than'water)'and'discard'appropriately''this'will'be'done'for'you'by'a'demonstrator'in'the'fumehood'using'a'glass'Pasteur'pipette'(chloroform'will'dissolve'plastic'Pasteur'pipettes).'The'upper'aqueous'layer'contains'your'nucleic'acid.'

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After'the'chloroform'has'been'removed,'cap'the'lid'tightly'and'spin'in'the'bench'centrifuge'for'10'minutes'at'4500g'at'room'temperature.''

Use'a'P1000'to'take'only'4mL'of'the'clear'top'aqueous'layer'and'transfer'to'a'new'screwZcapped'tube.$

2.$Removal$of$protein$ Add'6.95mL'5M'Na'perchlorate'(!"#$!!)'and'mix'well'by'inversion'(try'not'

to'create'bubbles).'The'high'ionic'strength'disrupts'the'DNA'protein'interactions'and'helps'to'remove'the'proteins'from'the'nucleic'acid.''

Add'an'equal'volume'(34.75mL)'of'chloroform:isoamyl'solution'(24:1)'and'firmly'screw'down'the'lid.'Chloroform'is'nasty'stuff'(carcinogenic'etc.)'and'is'stored'in'the'fumehood.'A'demonstrator'will'do'this'addition'for'you'in'the'fumehood.'Only'take'the'tube'out'once'the'lid'is'tightly'screwed'on.'

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Invert'the'tube'by'hand'continuously'for'5Z10'minutes''wear'gloves'and'ensure'the'lid'remains'tightly'sealed'onto'the'tube!'This'mixing'step'is'more'vigorous'than'using'a'mechanical'shaker.''

At'the'end'of'the'mixing'step,'allow'the'tube'contents'to'settle'(~5Z10'minutes).'Remove'the'bottom'chloroform'layer'(chloroform'is'denser'than'water)'and'discard'appropriately''this'will'be'done'for'you'by'a'demonstrator'in'the'fumehood'using'a'glass'Pasteur'pipette'(chloroform'will'dissolve'plastic'Pasteur'pipettes).'The'upper'aqueous'layer'contains'your'nucleic'acid.'

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After'the'chloroform'has'been'removed,'cap'the'lid'tightly'and'spin'in'the'bench'centrifuge'for'10'minutes'at'4500g'at'room'temperature.''

Use'a'P1000'to'take'only'4mL'of'the'clear'top'aqueous'layer'and'transfer'to'a'new'screwZcapped'tube.$

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3.$Recovering$the$Nucleic$Acid$$

Dispense'8mL'of'iceZcold'100%'ethanol'into'another'new'screwZcapped'tube.''

VERY'carefully'layer'2'volumes'of'this'cold'100%'ethanol'on'top'of'the'aqueous'layer.'As'you'add'the'ethanol'you'should'notice'a'white'gelatinous'precipitate'forming'at'the'interface.'This'is'your'nucleic'acid.'At'this'point,'do'NOT'mix'the'contents'of'the'tube!''

Collect'this'stringy'precipitate'with'a'glass'rod'by'twirling'it'(not'stirring!)'at'the'interface.'This'process'takes'some'time'to'ensure'a'good'yield'of'high'quality'nucleic'acid.''

Dispense'8mL'of'iceZcold'70%'ethanol'into'the'tube'that'you'used'for'100%'ethanol.''

After'you'have'wrapped'the'precipitate'around'the'glass'rod,'allow'the'excess'solvent'to'drain'off'and'dip'the'rod'in'the'tube'containing'70%'ethanol.'This'time'press'out'the'excess'solvent'and'then'drop'the'glass'rod'containing'your'lovely'clean'nucleic'acid'into'a'new'microcentrifuge'tube'containing'1mL'T.E.'(10mM'Tris,'1mM'EDTA,'pH'8).'Allow'the'precipitated'nucleic'acid'to'reZhydrate'and'dissolve'in'the'TE.'Put'the'glass'rod'back'into'the'original'tube'with'the'ethanol'and'aqueous'layer'and'twirl'the'DNA'again.$

3.$Recovering$the$Nucleic$Acid$$

Dispense'8mL'of'iceZcold'100%'ethanol'into'another'new'screwZcapped'tube.''

VERY'carefully'layer'2'volumes'of'this'cold'100%'ethanol'on'top'of'the'aqueous'layer.'As'you'add'the'ethanol'you'should'notice'a'white'gelatinous'precipitate'forming'at'the'interface.'This'is'your'nucleic'acid.'At'this'point,'do'NOT'mix'the'contents'of'the'tube!''

Collect'this'stringy'precipitate'with'a'glass'rod'by'twirling'it'(not'stirring!)'at'the'interface.'This'process'takes'some'time'to'ensure'a'good'yield'of'high'quality'nucleic'acid.''

Dispense'8mL'of'iceZcold'70%'ethanol'into'the'tube'that'you'used'for'100%'ethanol.''

After'you'have'wrapped'the'precipitate'around'the'glass'rod,'allow'the'excess'solvent'to'drain'off'and'dip'the'rod'in'the'tube'containing'70%'ethanol.'This'time'press'out'the'excess'solvent'and'then'drop'the'glass'rod'containing'your'lovely'clean'nucleic'acid'into'a'new'microcentrifuge'tube'containing'5mL'T.E.'(10mM'Tris,'1mM'EDTA,'pH'8).'Allow'the'precipitated'nucleic'acid'to'reZhydrate'and'dissolve'in'the'TE.'Put'the'glass'rod'back'into'the'original'tube'with'the'ethanol'and'aqueous'layer'and'twirl'the'DNA'again.$ Thin

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4.$Assessing$the$yield$and$quality$of$nucleic$acid$'

You'will'need'to'dilute'the'nucleic'acid'solution'by'~'20'fold'to'obtain'an'absorbance'in'the'working'range'for'the'spectrophotometer.'Remembering'that'the'quartz'cuvettes'required'to'measure'absorbances'at'wavelengths'



Report$item$4:$Explain$why$these$changes$would$alter$the$protocol$but$not$the$method.$

The'protocol'is'a'set'of'very'lab'specific'instructions'used'to'perform'the'isolation'procedure.'The'protocol'contains'items'such'as'specific'test'tubes,'and'exact'volumes'based'off'the'stock'solutions'and'items'within'a'specific'laboratory.'The'method'however'is'a'description'of'the'process'followed'to'obtain'a'particular'set'of'results;'this'is'not'lab'specific'and'does'not'include'either'items'such'as'the'particular'type'of'test'tube'or'exact'volumes,'instead'it'contains'lab'independent'measures'such'as'the'concentration'of'particular'chemicals'and'reagents.'

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The'difference'between'the'method'and'protocols'is'best'demonstrated'through'comparing'them'in'light'of'the'addition'of'SDS'within'the'isolation'practical.'

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Method' ''A'culture'of'E.'coli'(~0.1'g'wet'weight/mL)'suspended'on'0.1M'NaCl,'10'mM'EDTA,'pH'8'was'incubated'with'2%(w/v)'sodium'dodecyl'sulphate'(SDS)''Protocol'1:'5mL'culture'of'E.'coli,'25%(w/v)'stock'solution'of'SDS''Add'0.4mL'25%(w/v)'SDS'to'the'suspension''!"#$!!!!!"#!"'' !! = 0.25!!!!!! = 0.02!!!!!! = !!!"!!!!!! = 5 + ! !!"'!!!!! = !!!!!0.25! = 0.02 5 + ! !0.25! = 0.1 + 0.02!!0.25! 0.02! = 0.1!! 0.23 = 0.1!! = 0.4!"!!!!' != 0.4!"!!!"!!"!!!"!!"#$!!"!!"#$%!!!!"#!$#%&'%("#!!"!2%(!/!!)''Protocol'2:'25mL'culture'of'E.'coli,'20%(w/v)'stock'solution'of'SDS''Add'2.5mL'20%(w/v)'SDS'to'the'suspension'' !! = 0.20!!!!!! = 0.02!!!!!! = !!!"!!!!!! = 25 + ! !!"'!!!!! = !!!!!0.20! = 0.02 25 + ! !0.20! = 0.5 + 0.02!!0.20! 0.02! = 0.5!! 0.18 = 0.5!! = 2.8!"!!!!' != 2.8!"!!!"!!"!!!"!!"#$!!"!!"#$%&&!!"#!$#%&'%("#!!"!2%(!/!!)''

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9052_dna Isolation Report Complete