LabManual
AcceleratedBiology
©2011eScienceLabs,LLC
Allrightsreserved
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TableofContents
Lab1:DiffusionandOsmosis
Lab2:EnzymeCatalysis
Lab3:MitosisandMeiosis
Lab4:PlantPigmentsandPhotosynthesis
Lab5:Respiration
Lab6:MolecularBiology
Lab7:GeneticsofOrganisms
Lab8:PopulationGeneticsandEvolution
Lab9:Transpiration
Lab10:PhysiologyoftheCirculatorySystem
Lab11:AnimalBehavior
Lab12:DissolvedOxygenandAquaticPrimaryProductivity
……‡Ž‡”ƒ–‡†‹‘Ž‘‰›
MolecularBiology
APBIOLOGY
Lab6:MolecularBiology
Conceptscovered
9‡•–”‹…–‹‘‡†‘—…Ž‡ƒ•‡•
9
‡Ž‡Ž‡…–”‘’Š‘”‡•‹•
9ƒ…–‡”‹ƒŽ–”ƒ•ˆ‘”ƒ–‹‘
9‘’‡–‡–…‡ŽŽ’”‡’ƒ”ƒ–‹‘
9‰‹‡‡”‹‰’Žƒ•‹†•
9Žƒ•‹†˜‡…–‘”•
9–‹„‹‘–‹…”‡•‹•–ƒ…‡
9
‡‡–‹…‡‰‹‡‡”‹‰
Š‡ƒ„‹Ž‹–›–‘‹•‘Žƒ–‡ƒ†ƒ‹’—Žƒ–‡‰‡‡–‹…ƒ–‡”‹ƒŽ™ƒ•‘‡‘ˆ–Š‡‘•–’‘™‡”ˆ—Ž…Šƒ‰‡•‹–Š‡
ˆ‹‡Ž†‘ˆ‘Ž‡…—Žƒ”„‹‘Ž‘‰›‹–Š‡Žƒ–‡ʹͲ–Š…‡–—”›Ǥ‘†ƒ›ǡ–Š‹•‘™Ž‡†‰‡‘ˆ–Š‡•–”—…–—”‡‘ˆ
ƒ†‹–•…Š‡‹…ƒŽ’”‘’‡”–‹‡•Šƒ•‡ƒ„Ž‡†•…‹‡–‹•–•–‘•–—†›ǡ‹†‡–‹ˆ›ǡƒ†–”ƒ•ˆ‘”–‘
‹’”‘˜‡Ž‹ˆ‡Ǥ
…ƒ„‡—•‡†‹ƒ˜ƒ”‹‡–›‘ˆ†‹ˆˆ‡”‡–
™ƒ›•ǡ„—–ˆ‹”•–‹–—•–„‡”‡‘˜‡†ˆ”‘–Š‡
…‡ŽŽǤ…ƒ„‡‹•‘Žƒ–‡†ˆ”‘…‡ŽŽ•™‹–Š
…‘‘…Š‡‹…ƒŽ’”‘…‡†—”‡•Ǥ‡ŽŽ•…ƒ„‡
‘’‡‡†—’ƒ†•‡’ƒ”ƒ–‡†ˆ”‘‘–Š‡”
’ƒ”–•„ƒ•‡†‘‹–••‘Ž—„‹Ž‹–›Ǥ…‡‹•
”‡‘˜‡†ˆ”‘–Š‡…‡ŽŽǡ‹–‹•‘ˆ–‡—•‡ˆ—Ž–‘
ˆ”ƒ‰‡––Š‡‹–‘ƒƒ‰‡ƒ„Ž‡•‹œ‡•
–Šƒ–…ƒ„‡ƒƒŽ›œ‡†„›•…‹‡–‹•–•ǤŠ‹•…ƒ
„‡ƒ…Š‹‡˜‡†™‹–Š–Š‡—•‡‘ˆ”‡•–”‹…–‹‘
‡œ›‡•ǡ™Š‹…Šƒ”‡•’‡…‹ƒŽ‹œ‡†’”‘–‡‹•
–Šƒ–…Ž‡ƒ˜‡ǤŠ‡•‡‡œ›‡•ƒ”‡…ƒŽŽ‡†
restrictionendonucleasesǡ‡œ›‡•–Šƒ–
ˆ”ƒ‰‡–‹ƒŽ‘•–‡˜‡”›‘”‰ƒ‹•ƒ–
•’‡…‹ˆ‹…–ƒ”‰‡–•‡“—‡…‡•Ǥ
›’‹…ƒŽŽ›ǡ”‡•–”‹…–‹‘‡†‘—…Ž‡ƒ•‡•ƒ”‡
—•‡†–‘…—–‹–‘‹†‹˜‹†—ƒŽ‰‡‡•Ǥ
Š‡”‡ƒ”‡†‹ˆˆ‡”‡–˜‡”•‹‘•‘ˆ–Š‡•‡
‡œ›‡•–Šƒ–…ƒ„‡—•‡†–‘–ƒ”‰‡–
†‹ˆˆ‡”‡–Ž‘…‹–‘ƒ……‘’Ž‹•Š‰‡‡Ǧ•’‡…‹ˆ‹…
‹•‘Žƒ–‹‘•ǤŠ‡•‡‡œ›‡•…ƒŠƒ˜‡•‡˜‡”ƒŽ Figure6.1Restrictionendonucleasescansnip
DNAintwoways,leavingeitherbluntendsor
‘†‡•‘ˆƒ…–‹‘Ǥ‘‡”‡•–”‹…–‹‘
stickyends.
‡œ›‡•†‹‰‡•–„‘–Š•–”ƒ†•‘ˆƒ––Š‡
•ƒ‡’‘•‹–‹‘ǡ”‡•—Ž–‹‰‹„Ž—–‡†•‘
–Š‡ˆ”ƒ‰‡–•Ǥ–Š‡”‡†‘—…Ž‡ƒ•‡•…Ž‡ƒ˜‡‡ƒ…Š•–”ƒ†‹†‹ˆˆ‡”‡–Ž‘…ƒ–‹‘•ǡ™Š‹…Š
’”‘†—…‡••–‹…›‡†•ȋ‘˜‡”Šƒ‰•ȌǤ‘”†‡”–‘•‡…—”‡ƒ’”‘’‡”ˆ‹–„‡–™‡‡–Š‡‹•‡”–‡†‰‡‡ƒ†
–Š‡’Žƒ•‹†ǡ•…‹‡–‹•–•—•–…Š‘‘•‡‡œ›‡•–Šƒ–ƒ‡–Š‡•ƒ‡–›’‡‘ˆ…—–‹–Š‡†‘‘”‘”‰ƒ‹•
ƒ•–Š‡”‡…‹’‹‡–ȏȋ•–‹…›–‘•–‹…›ƒ†„Ž—––‘„Ž—–ȋ‹‰—”‡͸ǤͳȌȐǤ
MolecularBiology
…‡ƒ•‡‰‡–‘ˆ‹•‘„–ƒ‹‡†ǡƒ—„‡”‘ˆ†‹ˆˆ‡”‡–’”ƒ…–‹…‡•…ƒ„‡—•‡†–‘•–—†›‹–Ǥ
Electrophoresis‹•ƒ‘Ž‡…—Žƒ”–‡…Š‹“—‡–Šƒ–—•‡•ƒ‡Ž‡…–”‹……—””‡––‘•‡’ƒ”ƒ–‡
ƒ…”‘‘Ž‡…—Ž‡•ȋ‹Ǥ‡Ǥ•‡‰‡–•ǡ’”‘–‡‹•ǡ‘Ž‹‰‘—…Ž‡‘–‹†‡•Ȍ‘ˆ˜ƒ”›‹‰•‹œ‡•–Š”‘—‰Šƒ
’‡”‡ƒ„Ž‡‰‡Žȋ‹‰—”‡͸ǤʹȌǤŠ‹•–‡…Š‹“—‡–ƒ‡•ƒ†˜ƒ–ƒ‰‡‘ˆ–Š‡…Šƒ”‰‡†‘Ž‡…—Ž‡•„›—•‹‰ƒ
‡Ž‡…–”‹…ˆ‹‡Ž†–‘•‡’ƒ”ƒ–‡–Š‡‘Ž‡…—Ž‡•„ƒ•‡†‘•‹œ‡ǤŠ‡‰‡Ž‹•ƒ–Š”‡‡Ǧ†‹‡•‹‘ƒŽƒ–”‹š™‹–Šƒ
…Š‘•‡’‘”‘•‹–›ƒ†…‘’‘•‹–‹‘–‘…ƒ’–—”‡•’‡…‹ˆ‹…•‹œ‡•‘ˆ–Š‡‘Ž‡…—Ž‡•–ƒ”‰‡–‡†ǤŠ‡‰‡ŽŠƒ•ƒ
•’‡…‹ƒŽŽ‘ƒ†‹‰œ‘‡ǡ…‘–ƒ‹‹‰
™‡ŽŽ•–Šƒ–•ƒ’Ž‡•‘ˆǡǡ‘”
’”‘–‡‹•…ƒ„‡–”ƒ•ˆ‡””‡†–‘Ǥ
‘ƒ†‹‰ƒ‡Ž‡…–”‘’Š‘”‡•‹•‰‡Ž™‹–Š
–Š‡•ƒ’Ž‡”‡“—‹”‡•’ƒ–‹‡…‡ƒ†
’”ƒ…–‹…‡ǤŠ‡•ƒ’Ž‡—•–„‡
…ƒ”‡ˆ—ŽŽ›ƒ†•Ž‘™Ž›’‹’‡––‡†‹–‘
–Š‡™‡ŽŽ•…”‡ƒ–‡†ƒ––Š‡–‘’‘ˆ–Š‡
‰‡ŽǤŠ‡ƒ‡Ž‡…–”‹……—””‡–‹•
ƒ’’Ž‹‡†–‘–Š‡‰‡Žǡ–Š‡‘Ž‡…—Ž‡•™‹ŽŽ
–”ƒ˜‡Ž–Š”‘—‰Š–Š‡‰‡Žƒ–†‹ˆˆ‡”‡–
”ƒ–‡•–‘™ƒ”†–Š‡…ƒ–Š‘†‡‘ˆ‘’’‘•‹–‡
…Šƒ”‰‡ǤŠ‡”ƒ–‡‹•†‡–‡”‹‡†„›
ƒ••ǡ…Šƒ”‰‡ǡ†‡•‹–›‘ˆ–Š‡‰‡Žǡƒ†
Figure6.2Fillingthewellsofanelectrophoresisgel •–”‡‰–Š‘ˆ–Š‡‡Ž‡…–”‹…ˆ‹‡Ž†Ǥ•–Š‡
•ƒ’Ž‡‹•ˆ‘”…‡†–Š”‘—‰Š–Š‡‰‡Žǡ
…‘’‘‡–•ƒ”‡•‡’ƒ”ƒ–‡†‹–‘„ƒ†•†‘™–Š‡Ž‡‰–Š‘ˆ–Š‡‰‡ŽǤƒ”‰‡”‘Ž‡…—Ž‡•™‹ŽŽ‘˜‡
‘”‡•Ž‘™Ž›–Š”‘—‰Š–Š‡‰‡ŽǤŠ‡”‡•—Ž–‹•†‹•–‹…–„ƒ†•ȋ‹‰—”‡͸Ǥ͵Ȍ–Šƒ–…ƒ„‡…‘””‡Žƒ–‡†–‘ƒ
‘Ž‡…—Žƒ”ƒ”‡”–‘†‡–‡”‹‡–Š‡‘Ž‡…—Žƒ”™‡‹‰Š–‘ˆ–Š‡‘Ž‡…—Ž‡•‹‡ƒ…Š„ƒ†ǡ–Š—•
’”‘˜‹†‹‰ƒ’‘•‹–‹˜‡‹†‡–‹ˆ‹…ƒ–‹‘‘ˆ‡ƒ…Š…‘’‘‡–ǤŽ‡…–”‘’Š‘”‡•‹•…ƒ„‡—•‡†–‘…‘’ƒ”‡
‰‡‘‡•‘ˆ†‹ˆˆ‡”‡–‘”‰ƒ‹••‘”‹†‹˜‹†—ƒŽ•ǡƒ•™‡ŽŽƒ•–‘Ž‘…ƒ–‡ƒ†‹†‡–‹ˆ›ƒ•’‡…‹ˆ‹…‰‡‡‘ˆ
‹–‡”‡•–Ǥ
…‹‡–‹•–•…ƒƒŽ•‘Dz”‡ƒ†dz–Š‡
•‡“—‡…‡‘ˆ—…Ž‡‘–‹†‡•–Šƒ–
…‘•–‹–—–‡•–Š‡ˆ”ƒ‰‡–Ǥ…‡
–Š‡•‡“—‡…‡‹•‘™ǡ…ƒ„‡
‘†‹ˆ‹‡†ƒ†”‡Ǧ‹•‡”–‡†‹–‘–Š‡
•‘—”…‡ǡ‘”‡˜‡‹•‡”–‡†‹–‘ƒ‘–Š‡”
‘”‰ƒ‹•Ǥ‘Ž‡…—Ž‡•–Šƒ–…‘‡
ˆ”‘‘‡‘”‰ƒ‹•ƒ†ƒ”‡‹•‡”–‡†
‹–‘ƒ‘–Š‡”ƒ”‡…ƒŽŽ‡†recombinant
DNAǤ
Figure6.3AfluorescentdyethatbindsDNA,canbe
Žƒ•‹†•ƒ”‡•ƒŽŽǡ…‹”…—Žƒ”ǡ
usedtovisualizebandsafterthegelhasbeenrun.
‡š–”ƒǦ…Š”‘‘•‘ƒŽǡ’‹‡…‡•‘ˆ
ȋʹͲͲͲǦͳͲǡͲͲͲ„ƒ•‡’ƒ‹”•Ȍ–Šƒ–ƒ”‡ƒ–—”ƒŽŽ›ˆ‘—†‹„ƒ…–‡”‹ƒǤŠ‡›„‡…ƒ‡—•‡ˆ—Ž‘Ž‡…—Žƒ”
„‹‘Ž‘‰›–‘‘Ž•™Š‡‹–™ƒ•†‹•…‘˜‡”‡†–Šƒ–ƒˆ‘”‡‹‰‰‡‡…‘—Ž†„‡‹•‡”–‡†‹–‘–Š‡’Žƒ•‹†ƒ†
–Š‡„ƒ…–‡”‹ƒ™‹ŽŽ’”‘†—…‡–Š‡’”‘†—…–‘ˆ–Šƒ–‰‡‡ȋ‹‰—”‡͸ǤͶȌǤŠ‹•–‡…Š‹“—‡Šƒ•‡ƒ„Ž‡†Š—‰‡
ƒ†˜ƒ…‡•‹–Š‡ˆ‹‡Ž†•‘ˆ‘Ž‡…—Žƒ”„‹‘Ž‘‰›ƒ†‰‡‘‹…•Ǥ
MolecularBiology
—–ˆ‹”•–ǡ–Š‡„ƒ…–‡”‹ƒ—•–„‡ƒ†‡
…‘’‡–‡–‹‘”†‡”–‘’ƒ••–Š‡
Š›†”‘’Š‘„‹…ƒ…”‘••–Š‡…‡ŽŽǯ•
‡„”ƒ‡ǤŠ‹•‹•ƒ……‘’Ž‹•Š‡†„›
’‡”‡ƒ„Ž‹œ‹‰–Š‡…‡ŽŽ‡„”ƒ‡ǡ
™Š‹…Š…”‡ƒ–‡••ƒŽŽŠ‘Ž‡•–Š‡
…ƒ’ƒ••–Š”‘—‰ŠǤ
Figure6.4Scientistsareabletoengineerplasmidsso
theycontainageneofinterestfromanotherorganism.
ƒ…–‡”‹ƒƒ”‡ƒ„Ž‡–‘ƒ…“—‹”‡‰‡‡–‹…
‹ˆ‘”ƒ–‹‘–Š”‡‡†‹ˆˆ‡”‡–™ƒ›•ǣ
…‘Œ—‰ƒ–‹‘ǡ–”ƒ•†—…–‹‘ǡƒ†
–”ƒ•ˆ‘”ƒ–‹‘ǤConjugation‹•–Š‡
–”ƒ•ˆ‡”‘ˆ–Š”‘—‰Š†‹”‡…–
…‡ŽŽǦ–‘Ǧ…‡ŽŽ…‘–ƒ…–ǤTransduction‹•
–Š‡‘˜‡‡–‘ˆˆ”‘‘‡
„ƒ…–‡”‹—–‘ƒ‘–Š‡”„›ƒ˜‹”—•Ǥ
ƒ•–Ž›ǡtransformation”‡•—Ž–•ˆ”‘
–Š‡—’–ƒ‡ƒ†‡š’”‡••‹‘‘ˆˆ‘”‡‹‰
Ǥ
Figure6.5Incorporationofaforeigngenecanleadtocellswithnewcharacteristics.
MolecularBiology
‘”†‡”–‘…Ž‘‡ƒ‰‡‡ǡ‹–••‡“—‡…‡—•–„‡ƒ––ƒ…Š‡†–‘•‘‡‹†‘ˆ…ƒ””‹‡”ǡƒŽ•‘ƒ†‡‘ˆ
ǡ™Š‹…Š…ƒ–”ƒ•’‘”–‹–‹–‘–Š‡…‡ŽŽǤ—…Šƒ‘Ž‡…—Žƒ”–‘‘Ž‹•…ƒŽŽ‡†ƒ˜‡…–‘”ǡ™Š‹…ŠƒŽ•‘
…‘–ƒ‹•–Š‡‹ˆ‘”ƒ–‹‘–Šƒ–†‹”‡…–•–Š‡Š‘•–…‡ŽŽ–‘”‡’Ž‹…ƒ–‡–Š‡–”ƒ•ˆ‘”‡†•‡“—‡…‡ȋ‘”‹‰‹‘ˆ
”‡’Ž‹…ƒ–‹‘ǡ‘”oriȌǡƒ†…‘–ƒ‹•ƒ•‡Ž‡…–‹˜‡ƒ”‡”–Šƒ–ƒŽŽ‘™••…‹‡–‹•–•–‘–”ƒ…–Š‡˜‡…–‘”‹
–”ƒ•ˆ‘”‡†…‡ŽŽ•Ǥ
‡™ƒ›•…‹‡–‹•–•ƒ”‡ƒ„Ž‡–‘‹†‡–‹ˆ›™Š‹…Š…‡ŽŽ•Šƒ˜‡„‡‡–”ƒ•ˆ‘”‡†‹•–‘ƒ††ƒantibiotic
resistance‰‡‡–‘–Š‡’Žƒ•‹†•‡“—‡…‡Ǥ›…—Ž–—”‹‰…‡ŽŽ•‹ƒ–‹„‹‘–‹…•ǡ•…‹‡–‹•–•…ƒ‡•—”‡ƒŽŽ
‘ˆ–Š‡…‡ŽŽ•–Šƒ–”‡ƒ‹Šƒ˜‡–ƒ‡—’–Š‡’Žƒ•‹†…ƒ””›‹‰–Š‡”‡•‹•–ƒ…‡‰‡‡ǡƒ†…ƒŽ…—Žƒ–‡–Š‡
–”ƒ•ˆ‘”ƒ–‹‘‡ˆˆ‹…‹‡…›Ǥ‘–Š‡”‡–Š‘†‹•–‘—•‡ƒ•‡Ž‡…–‹˜‡ƒ”‡”–Šƒ–ƒ‡•–”ƒ•ˆ‘”‡†
…‡ŽŽ•ƒ†‹ˆˆ‡”‡–…‘Ž‘”ǡ‘”‡˜‡‰Ž‘™Ǩ
Š‡•‡‡–Š‘†‘Ž‘‰‹‡•ƒŽŽ‘™’”‡…‹•‡…‘–”‘Ž‘˜‡”–Š‡‹…‘”’‘”ƒ–‹‘‘ˆ’Žƒ•‹†•‹–‘…‡ŽŽ•ǤŽƒ•‹†•
ƒ›”‡ƒ‹‡š–”ƒǦ…Š”‘‘•‘ƒŽƒ†•‡ŽˆǦ”‡’Ž‹…ƒ–‡ǡ‘”–Š‡›…ƒ‹•‡”–‹–‘–Š‡‘ˆ–Š‡…‡ŽŽ
Šƒ”„‘”‹‰–Š‡’Žƒ•‹†ǤŠ”‘—‰Šƒƒ•–—–‡—†‡”•–ƒ†‹‰‘ˆ‰‡‡–‹…•ǡ’Žƒ•‹†•ǡƒ†”‡•–”‹…–‹‘
‡†‘—…Ž‡ƒ•‡•ǡ•…‹‡–‹•–•ƒ”‡ƒ„Ž‡–‘engineerplasmids–‘ƒ‡†–Š‡‘Ž‡…—Žƒ”’‘”–”ƒ‹–‘ˆ
‘”‰ƒ‹••ȋ‹‰—”‡͸ǤͷȌǤ
Pre-Laboratory Questions
ͳǤ ”ƒ•ˆ‘”ƒ–‹‘ǡ…‘Œ—‰ƒ–‹‘ǡƒ†–”ƒ•†—…–‹‘™‡”‡†‹•…‘˜‡”‡†‹–Š‡Žƒ„‘”ƒ–‘”›Ǥ‘™
‹’‘”–ƒ–ƒ”‡–Š‡•‡‡…Šƒ‹••‘ˆ‰‡‡–‹…”‡…‘„‹ƒ–‹‘‹ƒ–—”‡ǫ
ʹǤ •‡–Š‡‹–‡”‡––‘‹˜‡•–‹‰ƒ–‡ˆ‹‰‡”’”‹–‹‰ǤŠƒ–‹•–Š‹•’”‘…‡••ǡƒ†Š‘™‹•
‡Ž‡…–”‘’Š‘”‡•‹•—•‡†‹…”‹‹ƒŽˆ‘”‡•‹…•ǫ
͵Ǥ ‘™…ƒ’Žƒ•‹†•„‡—•‡†–‘ƒ—ˆƒ…–—”‡’”‘–‡‹••—…Šƒ•‹•—Ž‹ˆ‘”†‹ƒ„‡–‹…’ƒ–‹‡–•ǡ
‘”ƒ–‹„‹‘–‹…•ˆ‘”„ƒ…–‡”‹ƒŽ‹ˆ‡…–‹‘•ǫ
ͶǤ ‡•‡ƒ”…Šƒ‰‡‡–‹…†‹•‡ƒ•‡–Šƒ–ƒ›„‡…—”‡†–Š”‘—‰Š–Š‡—•‡‘ˆ‰‡‡–‹…‡‰‹‡‡”‹‰Ǥ
Šƒ––‡…Š‹“—‡•ƒ”‡„‡‹‰‡š’Ž‘”‡†–‘…‘””‡…––Š‡’”‘„Ž‡ǫ‘™†‘…‘…‡”•‘ˆ
†‡•‹‰‡”‡„”›‘••–‡ˆ”‘–Š‹•–‡…Š‘Ž‘‰›ǫȋUseadditionalpaperasnecessaryȌ
MolecularBiology
Experiment6.1:RestrictionEndonucleasesandElectrophoresis
Š‡‘„Œ‡…–‹˜‡‘ˆ–Š‹•Žƒ„‹•–‘—†‡”•–ƒ†–Š‡”‘Ž‡‘ˆ”‡•–”‹…–‹‘‡œ›‡•‹ˆ”ƒ‰‡–‹‰ƒ•–”ƒ†‘ˆ
ǡƒ†Š‘™–Š‘•‡ˆ”ƒ‰‡–•…ƒ„‡•‡’ƒ”ƒ–‡†—•‹‰‡Ž‡…–”‘’Š‘”‡•‹•Ǥ
Materials
ͻ‡†’‘’Ǧ‹–„‡ƒ†•
ͺ‡ŽŽ‘™’‘’Ǧ‹–„‡ƒ†•
͸
”‡‡’‘’Ǧ‹–„‡ƒ†•
ͳʹŽ—‡’‘’Ǧ‹–„‡ƒ†•
ȗ‘Ž‘”‡†’‡…‹Ž•
ȗ‘——•–’”‘˜‹†‡
Figure6.6
Procedure Table6.1
ͳǤ •‡–Š‡†‹ƒ‰”ƒƒ„‘˜‡ƒ•ƒ
”‡ˆ‡”‡…‡–‘…‘•–”—…–ƒ
•–”‹‰‘ˆ’‘’Ǧ‹–„‡ƒ†•™‹–Š
–Š‡•ƒ‡…‘Ž‘”’ƒ––‡”
ȋ‹‰—”‡͸Ǥ͸ȌǤ
ʹǤ •‡Ǯ‡œ›‡ͳǯ–‘ˆ”ƒ‰‡–
–Š‡•–”ƒ†‘ˆȋ’‘’Ǧ‹–
„‡ƒ†•Ȍ„ƒ•‡†‘–Š‡
Ž‹‰ƒ–‹‘‹ˆ‘”ƒ–‹‘
’”‘˜‹†‡†‹ƒ„Ž‡͸ǤͳǤ
͵Ǥ •‡–Š‡…‘Ž‘”‡†’‡…‹Ž•–‘
†”ƒ™–Š‡ˆ”ƒ‰‡–•‹œ‡•‹
–Š‡ƒ’’”‘’”‹ƒ–‡…‡ŽŽ‘ˆ
ƒ„Ž‡͸ǤͳǤ
ͶǤ ‹‡—’–Š‡ˆ”ƒ‰‡–•ƒ•
–Š‡›™‘—Ž†•‡’ƒ”ƒ–‡‹ˆ”—
–Š”‘—‰Šƒ‡Ž‡…–”‘’Š‘”‡•‹•
‰‡ŽǤ•‡–Š‡…‘Ž‘”‡†
’‡…‹Ž•–‘†”ƒ™–Š‡‹–Š‡
ƒ’’”‘’”‹ƒ–‡…‡ŽŽ‘ˆƒ„Ž‡
͸ǤͳǤ
MolecularBiology
ͷǤ‡’‡ƒ––Š‹•’”‘…‡†—”‡ˆ‘”‡ƒ…Š‡œ›‡(Note:Enzyme2and3meansthatfirst,enzyme2
fragmentstheDNA,thenenzyme3cutsthefragmentsmadebyenzyme2).
͸Ǥ‘‰‹–‘–Š‡‡…‹‡…‡ƒ„•™‡„•‹–‡ȋ™™™Ǥ‡•…‹‡…‡Žƒ„•Ǥ…‘Ȍƒ†’‡”ˆ‘”‡Ž‡…–”‘’Š‘”‡•‹•
™‹–Š–Š‡˜‹”–—ƒŽŽƒ„—†‡”–Š‡†˜ƒ…‡†Žƒ…‡‡–‹‘Ž‘‰›‘Ž‡…—Žƒ”‹‘Ž‘‰›•‡…–‹‘Ǥ
Questions
ͳǤ ‘™…ƒ„‡ˆ”ƒ‰‡–‡†‹–‘˜‡”›•’‡…‹ˆ‹…•‡…–‹‘•ǫ
ʹǤ Š‡”‡†‘”‡•–”‹…–‹‘‡œ›‡•…‘‡ˆ”‘ǫŠƒ–‹•–Š‡‹”ˆ—…–‹‘‹ƒ–—”‡ǫ
͵Ǥ ‘™†‘‘Ž‡…—Ž‡•‘ˆ˜ƒ”›‹‰•‹œ‡••‡’ƒ”ƒ–‡‹‡Ž‡…–”‘’Š‘”‡•‹•ǫŠƒ–‹•–Š‡’—”’‘•‡‘ˆ
–Š‡‰‡ŽǫŠƒ–ƒ„‘—––Š‡‡Ž‡…–”‹…‹–›ǫ
ͶǤ ˜‡•–‹‰ƒ–‡‘‡™ƒ›‹™Š‹…Š‡Ž‡…–”‘’Š‘”‡•‹•‹•—•‡†‹‡†‹…‹‡–‘†ƒ›Ǥ”‹–‡‘‡‘”
–™‘’ƒ”ƒ‰”ƒ’Š•–‘‡š’Žƒ‹›‘—”ˆ‹†‹‰•Ǥ
MolecularBiology
Lab6.2:Electrophoresis
–Š‹•Žƒ„ǡ›‘—™‹ŽŽ’”‡’ƒ”‡ƒƒ‰ƒ”‘•‡‰‡Žƒ†—•‡ƒ‡Ž‡…–”‹……—””‡––‘•‡’ƒ”ƒ–‡†›‡•‘ˆ†‹ˆˆ‡”‡–
‘Ž‡…—Žƒ”™‡‹‰Š–•ƒ†…Šƒ”‰‡•Ǥ‘—™‹ŽŽƒŽ•‘Šƒ˜‡–‘ƒƒŽ›œ‡–™‘†›‡•‘ˆ—‘™…‘’‘•‹–‹‘
ƒ††‡–‡”‹‡™Šƒ–‹š–—”‡–Š‡›ƒ”‡„ƒ•‡†‘”‡•—Ž–•ˆ”‘›‘—”‘™•ƒ’Ž‡•Ǥ
‡ˆ‹‡ˆ‘”–Š‹•‡š’‡”‹‡–ǣ
›’‘–Š‡•‹•ǣ
‘–”‘Žȋ•Ȍǣ
†‡’‡†‡–ƒ”‹ƒ„Ž‡ȋ•Ȍǣ
‡’‡†‡–ƒ”‹ƒ„Ž‡ȋ•Ȍǣ
Materials
‹‹‰‡Ž‡Ž‡…–”‘’Š‘”‡•‹•…Šƒ„‡”
͸‘‘–Š…‘„
Ž‹‰ƒ–‘”…Ž‹’•
ͻƒ––‡”›
ȋͳȌͳͲͲŽ‡ƒ‡”
ƒ”„‘ˆƒ„”‹…
͸‹…”‘Ǧ’‹’‡––‡•
ͳͲ—ˆˆ‡”
ȗ‘‹Ž‹‰™ƒ–‡”„ƒ–Š‘”‹…”‘™ƒ˜‡‘˜‡
‡–Š›Ž‘”ƒ‰‡
‘…‡ƒ—
›Ž‡‡…›ƒ‘Ž
›”‘‹
‘™͓ͳ
‘™͓ʹ
ͲǤͺΨ‰ƒ”‘•‡
ȗƒ–‡”
—Ž‡”
ȗ‘——•–’”‘˜‹†‡
Note:Gelsmustbeanalyzedimmediatelyafterelectrophoresis.
Colorbandswilldiffuseandbleedovertime.
1. ‘’”‡’ƒ”‡ƒͳ•‘Ž—–‹‘‘ˆȋ”‹•Ǧ„‘”ƒ–‡Ȍǡ‹šͳͲŽ‘ˆ–Š‡ͳͲ™‹–ŠͻͲŽ
™ƒ–‡”‹ƒͳͲͲŽ„‡ƒ‡”ǤNote:Ifthe10Xsolutionhasprecipitated,placethebottleinahot
waterbathandswirluntilprecipitantdissolves.
MolecularBiology
2. ‘’”‡’ƒ”‡–Š‡ƒ‰ƒ”‘•‡ǡŽ‘‘•‡–Š‡…ƒ’‘–Š‡„‘––Ž‡‘ˆͲǤͺΨƒ‰ƒ”‘•‡ƒ†Š‡ƒ–‹–Š‡
‹…”‘™ƒ˜‡‘”„‘‹Ž‹‰™ƒ–‡”„ƒ–ŠǤ‘‹–‘”–Š‡„‘––Ž‡…Ž‘•‡Ž›ƒ†”‡‘˜‡–Š‡„‘––Ž‡ˆ”‘–Š‡
Š‡ƒ–‡˜‡”›ͳͷ•‡…‘†•ƒ†•™‹”Ž–Š‡…‘–‡–•–‘‹šǤNote:Theagarosewillbecomeclearwhen
melted,about40Ǧ45seconds.
͵Ǥ ‡‘˜‡–Š‡„‘––Ž‡ˆ”‘Š‡ƒ–ƒ†Ž‡–…‘‘Ž–‘ƒ•ƒˆ‡Šƒ†Ž‹‰–‡’‡”ƒ–—”‡ȋ‹–•Š‘—Ž†•–‹ŽŽˆ‡‡Ž
™ƒ”̱͸ͲιȌǤ
ͶǤ •‡”––Š‡…‘„‹–‘–Š‡•Ž‘–•‹–Š‡‰‡Ž…‘’ƒ”–‡––‘‘Ž†–Š‡™‡ŽŽ•™Š‡–Š‡‰‡Ž‹•ˆ‘”‡†Ǥ
5. ƒ”‡ˆ—ŽŽ›’‘—”ͳʹŽ‘ˆ–Š‡˜‹•…‘—•ƒ‰ƒ”‘•‡‹–‘–Š‡‰‡Ž…‘’ƒ”–‡–‘ˆ–Š‡‹‹Ǧ‰‡Ž…Šƒ„‡”Ǥ
Š‡…‘–ƒ‹‡”Š‘Ž†•‘”‡ƒ‰ƒ”‘•‡–Šƒ™Šƒ–‹•‡‡†‡†–‘ˆ‹ŽŽ–Š‡…Šƒ„‡”ǡ•‘†‘‘–’‘—”ƒŽŽ
–Š‡ƒ‰ƒ”‘•‡‹ǡ‘Ž›ˆ‹ŽŽ‹„‡–™‡‡–Š‡–™‘”ƒ‹•‡†‰”‘‘˜‡•‘–Š‡„‘––‘‘ˆ–Š‡…Šƒ„‡”
ȋ‹‰—”‡͸Ǥ͹ȌǤHint:Thegroovesarenoticeablefromaboveandfromtheside,about1/4inchfrom
thetopandbottomofthechamber.Ifyouprefer,itmaybehelpfultobeeyelevelwiththe
chamberwhenpouringtheagarose.Whenlookingstraighton,fromalateralview,youcansee
theheightofthegroovesandwheretostopwhenpouringtheagarose.
͸Ǥ Š‹Ž‡–Š‡‰‡Ž‹••–‹ŽŽŽ‹“—‹†ǡ‹–‹•‹’‘”–ƒ––‘”‡‘˜‡ƒ›ƒ‹”„—„„Ž‡•–Šƒ–ƒ›Šƒ˜‡„‡‡
‹–”‘†—…‡†™Š‹Ž‡’‘—”‹‰ǤHint:Useapipettetipto“poke”thebubblesormovethemtothe
side.
͹Ǥ …‡–Š‡‰‡ŽŠƒ••‘Ž‹†‹ˆ‹‡†ȋͳͲȂͳͷ‹—–‡•Ȍǡ’‘—”‡‘—‰Šͳ„—ˆˆ‡”–‘…‘’Ž‡–‡Ž›
•—„‡”‰‡–Š‡‰‡Žǡƒ„‘—–ʹͲŽǤ
ͺǤ ƒ”‡ˆ—ŽŽ›”‡‘˜‡–Š‡…‘„ǤHint:Youmayhavetosoftlyjigglethecomboutofthegel.
ͻǤ …‡–Š‡…‘„‹•”‡‘˜‡†›‘—™‹ŽŽ•‡‡•‹š–”‘—‰Š•ǡ‘”™‡ŽŽ•ǡ‹–Š‡‰‡ŽǤŠ‹•‹•™Š‡”‡›‘—™‹ŽŽ
’‹’‡––‡–Š‡•ƒ’Ž‡•ǤNote:Thedyeshavebeenmixedwithasucrosesolutiontoincreasetheir
densityandinsureyoursamplewillfallgentlyintothewell.However,itisstillimportanttorun
thegelrelativelyquicklysothedyesdonotdiffuseoutofthewells.
ͳͲǤ—––Š‡…ƒ”„‘ˆƒ„”‹…–‘ƒ„‘—–ͳǤͷ•“Ǥ‹…Šǡ‘‡ˆ‘”–Š‡”‡†…Ž‹’ƒ†‘‡ˆ‘”–Š‡„Žƒ……Ž‹’Ǥ
‘Ž†–Š‡…ƒ”„‘ˆƒ„”‹…•‘–Šƒ–›‘—…ƒ†”ƒ’‡‹–‘˜‡”–Š‡–‘’ƒ†„‘––‘‡†‰‡•‘ˆ–Š‡‰‡Ž
…Šƒ„‡”ǤŠ‡ˆƒ„”‹…•Š‘—Ž†„‡•—„‡”‰‡†‹–Š‡ǤŽƒ’–Š‡„Žƒ…ƒŽŽ‹‰ƒ–‘”…Ž‹’–‘–Š‡
–‘’ȋ…Ž‘•‡•––‘™Š‡”‡›‘—Ž‘ƒ†‡†•ƒ’Ž‡•Ȍǡƒ†–Š‡”‡†…Ž‹’–‘–Š‡„‘––‘ȋ‹‰—”‡͸ǤͺȌǤ
•‡…ƒ—–‹‘™Š‡’‘—”‹‰
–Š‡‰‡Ž•‘–Šƒ–›‘—†‘‘–
‹–”‘†—…‡ƒ‹”„—„„Ž‡•ǡ
™Š‹…Š™‹ŽŽƒˆˆ‡…–Š‘™›‘—”
•ƒ’Ž‡•–”ƒ˜‡Ž–Š”‘—‰Š–Š‡
ƒ‰ƒ”‘•‡Ǥ
MolecularBiology
ͳͳǤ ǤŠƒ‡†‘™–Š‡†›‡ˆ”‘–Š‡–‘’‘ˆ‡ƒ…Š–—„‡•‘‹–…‘ŽŽ‡…–•ƒ––Š‡„‘––‘‘ˆ–Š‡–—„‡Ǥ
ͳʹǤ ‘ƒ†–Š‡†›‡•ƒ’Ž‡•ˆ”‘Ž‡ˆ––‘”‹‰Š–ˆ‘ŽŽ‘™‹‰–Š‡‘”†‡”Ž‹•–‡†„‡Ž‘™‹ƒ„Ž‡͸ǤʹǤ
Š‡Ž‘ƒ†‹‰–Š‡•ƒǦ
’Ž‡•ǡ–”›‘––‘•“—‡‡œ‡
ƒƒ‹”„—„„Ž‡‹–‘–Š‡
™‡ŽŽǤŠ‹•™‹ŽŽƒ‡–Š‡
†›‡…‘‡‘—–‘ˆ–Š‡™‡ŽŽǤ
ͳ͵Ǥ ‹–Šƒ•–‡ƒ†›Šƒ†ǡ’‹…—’ʹͲρŽ™‹–Š–Š‡‡‡†Ž‡Ǧ’‘‹–’‹’‡––‡ȋ‘—•Š‘—Ž†ˆ‹ŽŽ–Š‡ƒ””‘™
’‘”–‹‘‘ˆ–Š‡’‹’‡––‡–‘–Š‡„‘––‘‘ˆ–Š‡•“—‡‡œ‡„—„„Ž‡ǤŠ‹•‹•ƒ„‘—–͹Ȁͺ‹…ŠǤȌ
ͳͶǤ š’‡Žƒ›ƒ‹”–Šƒ–‹•ƒ––Š‡–‹’‘ˆ–Š‡’‹’‡––‡•‘–Šƒ–‹–‹•‘–‹–”‘†—…‡†‹–‘–Š‡™‡ŽŽǤ‡‘˜‡
‡š…‡••†›‡‘ˆˆ–Š‡‘—–•‹†‡‘ˆ–Š‡’‹’‡––‡•‘ƒ•–‘‘–‹–”‘†—…‡ƒ›‡š…‡••–‘–Š‡ͳǤ
ͳͷǤ —„‡”‰‡–Š‡–‹’‹–‘–Š‡„—ˆˆ‡”–ƒ”‰‡–‹‰–Š‡†‡•‹”‡†™‡ŽŽǡƒ†•Ž‘™Ž›†‹•’‡•‡–Š‡†›‡
•ƒ’Ž‡‹–‘–Š‡™‡ŽŽǤ
Figure6.7Connectingtheleadstothe
miniǦgelchamberandpowersupply
16. ‡’‡ƒ––Š‹•’”‘…‡••ˆ‘”‡ƒ…Š†›‡•ƒ’Ž‡ǡ‹…Ž—†‹‰–Š‡–™‘—‘™•ǤRemembertousea
cleanpipetteforeachsample!
ͳ͹Ǥ …‡ƒŽŽ•ƒ’Ž‡•Šƒ˜‡„‡‡Ž‘ƒ†‡†ǡƒ––ƒ…Š–Š‡…ƒ”„‘‡Ž‡…–”‘†‡•–‘‡‹–Š‡”‡†‘ˆ–Š‡‹‹Ǧ‰‡Ž
…Šƒ„‡”ƒ••Š‘™‹‹‰—”‡͸Ǥ͹Ǥ
MolecularBiology
ͳͳǤŠƒ‡†‘™–Š‡†›‡ˆ”‘–Š‡–‘’‘ˆ‡ƒ…Š–—„‡•‘‹–…‘ŽŽ‡…–•ƒ––Š‡„‘––‘‘ˆ–Š‡–—„‡Ǥ
ͳʹǤ‘ƒ†–Š‡†›‡•ƒ’Ž‡•ˆ”‘Ž‡ˆ––‘”‹‰Š–ˆ‘ŽŽ‘™‹‰–Š‡‘”†‡”Ž‹•–‡†„‡Ž‘™‹ƒ„Ž‡͸ǤͳǤ
ͳ͵Ǥ‹–Šƒ•–‡ƒ†›Šƒ†ǡ’‹…—’ʹͲρŽ™‹–Š–Š‡‡‡†Ž‡Ǧ’‘‹–’‹’‡––‡ȋ‘—•Š‘—Ž†ˆ‹ŽŽ–Š‡ƒ””‘™
’‘”–‹‘‘ˆ–Š‡’‹’‡––‡–‘–Š‡„‘––‘‘ˆ–Š‡•“—‡‡œ‡„—„„Ž‡ǤŠ‹•‹•ƒ„‘—–͹Ȁͺ‹…ŠǤȌ
ͳͶǤš’‡Žƒ›ƒ‹”–Šƒ–‹•ƒ––Š‡–‹’‘ˆ–Š‡’‹’‡––‡•‘–Šƒ–‹–‹•‘–‹–”‘†—…‡†‹–‘–Š‡™‡ŽŽǤ‡‘˜‡
‡š…‡••†›‡‘ˆˆ–Š‡‘—–•‹†‡‘ˆ–Š‡’‹’‡––‡•‘ƒ•–‘‘–‹–”‘†—…‡ƒ›‡š…‡••–‘–Š‡ͳǤ
ͳͷǤ—„‡”‰‡–Š‡–‹’‹–‘–Š‡„—ˆˆ‡”–ƒ”‰‡–‹‰–Š‡†‡•‹”‡†™‡ŽŽǡƒ†•Ž‘™Ž›†‹•’‡•‡–Š‡†›‡
•ƒ’Ž‡‹–‘–Š‡™‡ŽŽǤ
ͳ͸Ǥ‡’‡ƒ––Š‹•’”‘…‡••ˆ‘”‡ƒ…Š†›‡•ƒ’Ž‡ǡ‹…Ž—†‹‰–Š‡–™‘—‘™•ǤRemembertousea
cleanpipetteforeachsample!
ͳ͹Ǥ…‡ƒŽŽ•ƒ’Ž‡•Šƒ˜‡„‡‡Ž‘ƒ†‡†ǡƒ––ƒ…Š–Š‡…ƒ”„‘‡Ž‡…–”‘†‡•–‘‡‹–Š‡”‡†‘ˆ–Š‡‹‹Ǧ‰‡Ž
…Šƒ„‡”ƒ••Š‘™‹‹‰—”‡͸Ǥ͹Ǥ
ͳͺǤ‘‡…––Š‡ƒŽŽ‹‰ƒ–‘”…Ž‹’•–‘–Š‡‡Ž‡…–”‘†‡••‘–Šƒ––Š‡‡‰ƒ–‹˜‡ȋ„Žƒ…Ȍ‹•…‘‡…–‡†–‘–Š‡
‡†™Š‡”‡›‘—Ž‘ƒ†‡†–Š‡•ƒ’Ž‡•ǡƒ†–Š‡’‘•‹–‹˜‡ȋ”‡†Ȍ‹•…‘‡…–‡†–‘–Š‡‘’’‘•‹–‡‡†Ǥ
ͳͻǤ––ƒ…Š–Š‡‘–Š‡”‡†•‘ˆ–Š‡ƒŽŽ‹‰ƒ–‘”…Ž‹’•–‘–Š‡ͻ„ƒ––‡”›Ǥƒ–…Š…ƒ”‡ˆ—ŽŽ›ˆ‘”ˆ‹˜‡Š‘—”•Ǥ
Note:DONOTletthedyesmoveoffthegel!
ʹͲǤ…‡•‡’ƒ”ƒ–‹‘‘ˆ–Š‡†›‡•Šƒ•„‡‡ƒ…Š‹‡˜‡†ǡ†‹•…‘‡…––Š‡ƒŽŽ‹‰ƒ–‘”…Ž‹’•ˆ”‘–Š‡„ƒ––‡”›Ǥ
ʹͳǤ‡ƒ•—”‡–Š‡†‹•–ƒ…‡ˆ”‘–Š‡™‡ŽŽ•–‘–Š‡…‡–‡”‘ˆ–Š‡„ƒ†•ƒ†”‡…‘”†–Š‹•‡ƒ•—”‡‡–
‹ƒ„Ž‡͸ǤʹǤ
ʹʹǤ Ž•‘‹ƒ„Ž‡͸Ǥʹǡ”‡…‘”†–Š‡—„‡”‘ˆ„ƒ†•˜‹•‹„Ž‡ˆ‘”‡ƒ…Š†›‡•ƒ’Ž‡Ǥ
Table6.2
ElectrophoresisofDyeSamples
Well
Sample
#Bands
Migration(cm)
ͳ
›”‘‹
ʹ
‡–Š›Ž”ƒ‰‡
͵
‘…‡ƒ—
Ͷ
›Ž‡‡›ƒ‘Ž
ͷ
‘™ͳ
͸
‘™ʹ
Questions
ͳǤŠƒ–‹•‹‡ƒ…Š‘ˆ–Š‡—‘™•ƒ’Ž‡•ǫ
MolecularBiology
ʹǤŠ›‹•‡Ž‡…–”‹…ƒŽ…—””‡–‡…‡••ƒ”›–‘•‡’ƒ”ƒ–‡‘Ž‡…—Ž‡•—•‹‰‡Ž‡…–”‘’Š‘”‡•‹•ǫ
͵ǤŠ›‹•ƒ‰ƒ”‘•‡ƒƒ’’”‘’”‹ƒ–‡‡†‹—–‘—•‡ˆ‘”•‡’ƒ”ƒ–‹‰‘Ž‡…—Ž‡•ǫ‡•‡ƒ”…Šƒ‘–Š‡”
–›’‡‘ˆ‰‡Žƒ†’”‘˜‹†‡ƒ„”‹‡ˆ‡š’Žƒƒ–‹‘”‡‰ƒ”†‹‰™Š›‹–…‘—Ž†„‡—•‡†”ƒ–Š‡”–Šƒƒ
ƒ‰ƒ”‘•‡‰‡ŽǤ
ͶǤ Šƒ–‹•–Š‡…Šƒ”‰‡‘ˆ–Š‡•ƒ’Ž‡•ǫŠƒ–†‘›‘—–Š‹™‘—Ž†Šƒ’’‡‹ˆ–Š‡‘Ž‡…—Ž‡•™‡”‡
–Š‡‘’’‘•‹–‡…Šƒ”‰‡ǫ
ͷǤ ‘™‹•‡Ž‡…–”‘’Š‘”‡•‹••‹‹Žƒ”–‘ǡƒ††‹ˆˆ‡”‡–ˆ”‘…Š”‘ƒ–‘‰”ƒ’Š›ǫ
1500WestHampdenAvenue
Suite5ͲH
Sheridan,CO80110
303Ͳ741Ͳ0674‫ڄ‬888ͲESLͲKITS
www.eScienceLabs.com