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by: Alysha Quigley


Alysha Quigley
Virginia Commonwealth University
GPA 3.87


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Class Notes
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This 5 page Class Notes was uploaded by Alysha Quigley on Wednesday October 28, 2015. The Class Notes belongs to BIOL 300 at Virginia Commonwealth University taught by Staff in Fall. Since its upload, it has received 8 views. For similar materials see /class/230648/biol-300-virginia-commonwealth-university in Biology at Virginia Commonwealth University.

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Date Created: 10/28/15
Sequencing Laboraton Methods secure 8 October Lab Week of 22 October mm Sequencmg Overview DN DN therr3 rb quot t m dye Duhhg PCR mostly hahve hueleotrdes are addedto the DNA template However when a W wd beeause there is no 3 OHto whreh the hexthueleoude eah be attaehed Hehee the dddeoxy th solutroh 1 M r excites e dye orhputer program evaluates the resulhhg hrstograrh omuoreseehee peaks and eaus the hueleohdes m the order they passed through the vrewer Read the followrhg pages and PRIOR TO LEcrURErhsta11 Chrorhas and evaluate the sample data le ehhued R10L3ooseq brhquot mom r aha1ysrs data hahdhhg SW6 5mm DNA ete to be sequenced 5 TGACTTCGACAA Add T T539 DNA potymetaset dATP dGTP dCTP der pms wmtmg amounts or uorescean tapeted ddATP Id rr ddCTP ddTl P fragmenB 3t s r r a r eteoroptorests seii iie n usmg aserto V ofthe quot actwate he E temptate E uorescent A stmnd 5 Y at w A Y quotroteoooesaroa g E detectorto i orsmh r r oops r a SmaHer V quot fragmenB 5 339 Laboratory Methods Objective remove residual reagents from a PCRamplified segment of DNA and then perform a uorescent termination sequencing reaction and analyze the resulting data to determine quality of the run and homology of the sequence generated Retrieve the 5 uL aliquot of DNA that you archived two weeks ago for sequencing Set up on ice an ExosapTM reaction as follows this will remove primers and residual dNTPs 0 Add 2 uL of ExosapTM to your PCR tube containing 5 uL of PCR product 0 Mix by pipetting twice 0 Place in thermal cycler cover with heated lid and run EXOSAP program 15 min 37 C 15 min 80 C hold 4 C Set up on ice two master mixes for the class as follows where is the number of PCR products to be sequenced plus one e g if there are six reactions to be sequenced today then make sufficient master mix for seven reactions Forward Mix for n1 reactions Reverse Mix for n1 reactions X 05 uL FonNard primer X 05 uL Reverse primer x15uL H20 x15uL H20 X 2 uL ET X 2 uL ET Set up on ice individual sequencing reactions as follows 0 Label two separate PCR tubes with the rst and last initials of each lab partner combined with either F for forward reaction or R for the reverse reaction For example your tubes might read BBANF and BBANR Be sure to use your own initials and to record the code in your notebook 0 Combine 4 uL of forward sequencing master mix plus 2 uL of ExosapTMtreated DNA in the tube labeled F 0 Combine 4 uL of reverse sequencing master mix plus 2 uL of ExosapTMtreated DNA in the tube labeled R 0 Place in thermal cycler cover with heated lid and run MEGAOPT program 20 sec 95 C followed by 40 cycles of 20 sec 95 C 15 sec 50 C 1 min 60 C followed by hold 4 C Your instructor will collect completed sequencing reactions that will subsequently be purified using a MontageTM filter plate and then analyzed on an automated sequencing analyzer in the VCU Ecological Genetics Laboratory Your data will be posted on BlackBoard around 5 November as a file named with the code you created in lab and having the file extension bin You must download this file and a program called Chromas to analyze your data Continue with next section entitled Interpreting DNA Sequencing Results and Examining Sequence Homology for information on Chromas and interpreting your sequence Interpreting DNA Sequencing Results and Examining Sequence Homology Interpreting Sequencing Results The computer output for a sequencing run consists of chromatogram in our case your lenamebin le showing the observed DNA sequence Usually there is some ambiguity at various sites along the DNA sequence and the subprograms used to call the bases can t precisely determine what nucleotide is represented by either a broad peak or a set of overlaying peaks In such cases a letter other than A C G or T is recorded most commonly N An investigator should proof read each sequence to ensure that all ambiguous sites are correctly called Here is an example of ambiguity in sequence output TTTTCATCAATTG39GIATTTA T El 560 El 1 1L1 1L1ACG 1L1LJLGGZL1 1 1L1 EC39 I I I I HATE ATTGIGNATIT 1 it r quotIn 39J39nl l i I 5 I39l g M II q in ii l 1 ii I this ll it 1 I Notice that the spacing between the two black G peaks near the end of the sequence indicates the possibility that there might have been peaks for Ts in between but those red peaks were so low that the computer could not con dently call them Thus in proo ng the sequence you might determine that there is suf cient indication that the correct sequence is likely to be TTTTCATCAATTG L G L ATTTA Assignment The rst part of your assignment is to view and correct your own sequence as necessary To accomplish this you will install the chromatogram viewer called ChromasTM by clicking on the le called chromas23 lexe on the following web page httpwwwtechnelv inm com 39 html and following the directions to install Chromas to your computer Chromas should open automatically following the install procedure at which point you will open and view your chromatogram yourfilenamebin FROM INSIDE THE CHROMAS PROGRAM Examine your sequence from beginning to end by pulling the scroll bar Good sequence generally begins around base 20 and is represented by tall distinct peaks that have little overlap Poor reactions will result in low or multiple peaks as illustrated below very typical of beginning or end of a reaction At the beginning of your sequence place the cursor at an appropriate location demarking poor and good sequence then choose Edit Set Left Cutoff from the toolbar Proceed to the end of your sequence place the cursor where data are no longer informative and select Edit Set Right Cutoff BEFORE CLAss ON FRIDAY PLEASE USE SAMPLE FILE BIOL3OOSEQBIN To FAMILIARIZE YOURSELF WITH THE PROGRAM AND DATA ANALYSIS 2 q The second part of your assignment is to in l l Epnm ueib El E Zl pal i i i i eii r 933 quot quot Hl iSampleH7 inasezn our Hi s u an acnc cminncrpucrmannenannnnmcc can Beginning of Sequence Showing reasonable settings for Left and Right LJLJLJL A m tril l Ell mi Evian Help z W Pa 3 illlHJLi mm rm 7n sunr 5w Marni1mmunnwgcnrnqunrqinnnnn rEHIVFHGGVEI 581 i J End of sequence cutoff of poor sequence data Next check your sequence for ambiguity by searching for places where N is the base call instead of an A C G or T If you can confidently score the ambiguity hilite the N and overtype with the correct base For example in the case shown to the right the correct nucleotide was likely a T I prefer to overtype with a lowercase letter to distinguish which bases were called by the program and which were corrected by the operator When you are finished trimming and correcting the sequence proceed to File Export give the file a new name and click Export which will create a text file of the sequence with the extension seq The text data subsequently can be opened in Notepad or any word processing program Basic BLAST cm arm pi determine homology of the corrected DNA sequence In other words is it similar to any other published sequences and if so to what degree This procedure will be accomplished using BLAST a program supported by the National Center for Biotechnology Information N CBI First open your corrected sequence lenameseq Next go to the BLAST window on the NCBI site at httpwwwncbinlmnihgovBLAST In the middle section of this page select Nucleotide BLAST Switch to your sequence copy it and toggle back to paste your sequence in the Enter Query Seq box at the top of the BLAST page Choose Nucleotide collection as the Database Click the Blast radio button to submit your sequence data nuclenlids mm Ernlein um mm rtnmnnnnnnau V ananawu l l l we in mi Lnlerlivan Mm Linzinal m EmnranEiiinZIiuarvn lmnzszrzquot a ngram Select on mm m MAM m m idiscan gtnus quotsmug ci39ri rmmmimuq u can Him mm b mam St E You will receive a screen similar to this one where you should click on Formatting Options and then check to ensure that Alignment View is set to Pairwise it neutuumttuum a r m 5 uuuueu uuwmem i 1 Meal Stun in Register HiCBi BLASTDiaslniFormamng Results VGM39I39FIIIV45015 Formatting options Job Tl e lct8056 645 letters GMTFMV45D15 Searching Mon Oct 81316 36 2007 Mun Oct 8131537 2007 Request lD Status Submitted at Current time me SInCE Submission Thls page wlll be autumailcaliy updated in 8 seconds Your search results will be displayed in a new window in a few seconds or a few minutes depending on how many other folks are BLASTing their data showing any hits your sequence made The results will be color coded and annotated so that you can either click on the horizontal homology bars or you can simply scroll down the page Distribution of 100 Blast Hils on the Que Seguence color key for allgnment scores lt40 40 50 an ar 50200 gt200 Query l l l l l l l o 100 200 300 400 500 600 Links to other GenBank pages showing matching accessions Dzame tree magma Legend in 1nk cc anger resources El UnlEene E can Gene E 56 cure In Map Vlewer Sequences pmaumnq slqnxfxcant alignments lchck header m 50 columns Deszrintinn Arahldauslsthailana chmmusume 3 BAG mam uenumlcseuuence I Pseu nmanas aemulnasa class 1 Warm metaiiarbetariactamase ml 1 NA a 6 cl HEAAA a a 3 a p E alttah Links to subsequent sections on the current results page The bars show what places along your sequence are similar to other published sequences The colors indicate how many bases were involved in homology determination The scores indicate how much homology occurred for particular hits Clicking on a score or bar in the graphic representation will take you to the section of the results page that describes a sequence that is similar to yours Clicking on the Accession link at the beginning of any line will take you to


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