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This 7 page Class Notes was uploaded by AnnaClippinger on Monday September 28, 2015. The Class Notes belongs to BIOL-L211 2521 at Indiana University taught by Megan Dunn in Summer 2015. Since its upload, it has received 39 views. For similar materials see Molecular Biology in Biology at Indiana University.
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Date Created: 09/28/15
Heur dut Lecture 8 59p 2015 Last Time I Replication Nucleic Acid Basic Methods 39 Today Problem Set 1 Due Fl Eu ding Mglqh mtw t i Trombone Model QampA t Ch l 23 39TZCiLi 301quot Be Nucleic Acid Basic Methods iIello mothers hello father i moltcular Bi elo EN l Tm NE MJ Replication Initiation Next Time Come wleuemnhrt She want new V quotTi thing PEWTER Rewew SeSSIon optiona no parthlpathh pomts Exam on Wednesday from 79 pm in Ballarltine Hall 013 liiUlJit l Uttll Brlng pencil all 30 iviultli li Chili i 2 Pt techs Carrier an on will sinw i gmntn dmwudmnml I Suggestions OlfF nd l liUi MW 1 Class Attendance Come to class engagedfocused on L211 Take notes THROUGHOUT lecture 7 V a l Ask questions when something is not clear rm 2 l takean UP The 3 used ms cowl i 4 used 0n mowing mm It 2 on Tm lugqmq ttlnnl Pre and PostLecture Look over Iectu re notes Read reievant sections of textbook pay close attention to gures used in lecture Review TZT activities lECl lil39E hU TES Answer weeklyf discussion group questions without notes then go back and revise answer Review problem set Molecular Biology in the News Reading Assignments Read the articles several times On FUT lg Go over them with your classmates Exam Preparation Spend at least 3 4 hours per lecture Of ce Hour Attendance Participate in the open of ce hour sessions Come with speci c targeted questions T1T 3 Which D39Nli mmqu mum he lime m Run Entree trim The gum Will a is blwismm 39 39 x m 1 l WiP e mm Method to analyze DNA fragments from PCR Dr restriction digests l n um an l trlml charge W Wale frag marl ii bailed0n Chamlber tilled with buffer R ELM Ne SIZE quot MIPS M 90W Wpurail mn Agarose gel electrophoresis Power source 39 W M M melded DN SUmPIQS quot i Agarose gel Mall 2 NE tw QWCTWdQ W m 3970 5 l gamma Rewmm 39 wam i0 Dll39llflcl it to the WWW Side 5 Will me Tm negllll Si 3 Agarose gel electrophoresis mecmpims agarose gel eer v Electric current applied to the gel DNH freq MQ MYMWWK Omar i 39 in Site 39 d quot9 17 in 39 l i u 1 l i l h 7 I T H W 2 quotETquot i ilQFi t I Ha 4w 3 l V 39 7 7 W12 39quot39l39 1 I quot 1 39l I l i l l smellDNMregmentsmuve a lu herthmuhthe gelthan 1 large fragmenis Emu Heal mwe f S StFig 71 Viewed by placrrig the gel on ail ultraviolet light emitting box alum Bmmld glm iS Uifd DNA will be cl ermm r Grunge under UV Mm V E v r a r NLurqel 0M rmqrn em ll lilll l llII Illlllllllll ll ll39lll Illl ill ii Ill llll I S mailer D NH mg WM 4 llll ll fllllll l l E L a Ii llllllllllll ll t Rememmr WWN mumm Urn DNA sequemmg Fiddll lCl Cl mmum iii 7 clNTPS to N F l the 0M St ainnum 39mnrule f t 39in a hadm li has a mirage Sqntmmsr Pi A e 39rc can made thainterwn39mmqv Remember DNA synthesis occurs lbyjeining the 3 Dl i of one nucleotide to a 5 phosphate of another 39 at end of strand l WSW edited Domi Ill 5 v WV DNA 5e uencinf Chain termina n etho 39 r a V 43 q 9 R 939 SEVDQKlm Mgr PRI MBRH TE Include man of the same lPCR ingredients 7 m1 Tl UP Q Ch DOSE For each mainterminating nu den Cl only L timer rename ream an f LEDBE I leaetion either Bl C Di Frerene Example Reachion with Wquot guilt 7 Vin V v 5 quot lV iiiquot r El I I 311 in l I39I H Hare i1 a itempiale D a 1ive 3 5 e a 9 1 if ma nil unwridd un ld m themmlom me rem lithe 39 lengating chain win 39 DNAsynthesis IP is added 2 N 1 Th C FEHW l mnilu re N ilfl ljl 13 UVQ W63 1 Chain 3912 W Wmnj 1 mama frag ment Generates fragments of various g m OMB SWUN 1 H m0 the lengths which can be separated on gel Sinner r E Awarenu DNA sequencing Chainaterminaiing method ddMP slitrteN E Il lllllill wlllll Illll lllll lill aux Illi ill l E ll l ll l illllll llllllllll 3 IIIquot I I II III llllll mm llliIlM ll l lilililliillilliii llllllllllHllllll mm lll39lllllilllll llllll lulll li llliljilllill I I ll 1 M I II II 9 Tlhen repeat for all reactions using same template Each larie A separate reaction with a chain terw na ngrun ee de Fragments separate on gel according to size Reutlfmm summer me an W The 9 T0 d clum sequential E we first hue will MT E gimme mm 0 ml oi coverage e hum rmmull llq alylmihl doesrll I l l Ii gllm n D IlI Hll fl 139 us in Will El qm m39 DNA sequencing Chain terminating method automated Tl sseparates DNAs based on length shorter emerges rst Readeut looks just like thlsm Template TCTGATTACGGCATTCGGTT Primer 39AGAETAATGGCGTAAGCCAAEE AGACTAATGCCGTAAGCCA AGACTAATGGCGTAAGCC AGACTAATGCCGTAAGC AQACTAATGCCGTAAG AGACTAATGECGTAA AGACTAATQQCGTA AGACTAATEGCGT AeeeTAATGCEG AeAeTAATeeC A 393 o D H DHT I 9 HMO7P5quot A Synthesis wi n uorescent djdeoxynu c1eetidee Capillary BlECITDPhDIEEiS EC Nucleotide sequence mmm gelqu Few u Eh meme Elm Hl l Willquot mime Ml Slings r mum lg ll J Enlln l eell l l i bur can mm an we e HQUTECI Quit a Nu 5quot WM ll n m a miss dW nWWRHVMT Tm militate in 1m Uni xx f when c1 M i ll Willem mum MWN Q Tu a ll Em u5t ll 1 largest 5 lower In H m seamen N a l mmquot m with f loun l L Il We 05 Tm 0rd in rm lollies me lmm we ll 539 m PCR and sequencing Rem world applications Example F 39renrsics V V 7 7 men rm human mane 1 Remus V tumult f m emnwa INI vldu lS m MUM Exam lei Stud extinct animals I p v Biol m me NEW w th 5L M 0 I mum WW 390 U TIw Uli Id v w E535 E cf ar 39E We 531mm VEN my 1 M on munth m a PWMCGI summon15 MIT Enzymes that fact Hike molecular scissors far analysis 7 V 7 7 wr qlqtsn D N a a we ML mum as L39R S tH tiWn MW ck n2 nuclewm Fownd in Bacteria and Archaea Sal c i commercially puri ed New Ewan E nz q m iS Exa um pie 3 9 9 Recogniz sand cuts 5 GAATFC3 39 PW dum WW a mgquot Fig 125 quotstickyquot and 10 mm W Restriction endonucledses E newts Used in research labs to cut DNA into smaller pieces Can separate pieces on an agarpse gel Example linear piece of DNA with 6 copies pf39GAA I I C sequence Agarose gel electrophoresis separates fragments based on size 7 Run on E quoti J ilEmTSlteslill J l agarose gel a 7 397 l A decreasing size I A VI aglclnl E l39rlel n quwmts C 1r 7 Fig 73 0quot the Kennellion W731 m fragments 11 l l il l I l Uses for restriction endon ucieases Can be used for diagnostic tests Example Large pieces of DNA cut with restriction enzymes Run on agarese gel Compare restriction digest fragments bandin g attern on gel Q M awe muqhwdqsm ms on Mares lmilntm bE i39NEEH mes pf own ll llll ll 39Tlllill iilll M A 139 lil llll l lllll llll H E E E Sl ll M 12 l39l39 39u Uses for restriction end onueleeses 39 cut a m m mm H Um 1LT 0 an other Xh I Recipient Pla 39 id Recipient Plasmid Also Iused frequently in DNA donmg 11m produuS of PL a Raw reli m1 MEETS hm WNW M 0N Lquot 3 End of Exam 1 material 14
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