Preparation of insert

Preparation of insert - Cloning Strategy , I t / l_ l r\ /...

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Cloning Strategy , I t / E*RI+Hindrn \ / Gcthris \' r\ l_ l l-1 smallFngmmt I''ugoFngEcnt t AllGlbor. EoRI+Iilndlll
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fntroduction fta t.G.l.cto5t d.rct Subcloning .\ t The pBSKVectot nntp sryl2s5o sq.'z Scal25rF Pntl2416 ,V.el gB A{rllSlP ,(pl1657 S.cl 759. Pqt,lCn IF.$Hf.:5 jr,',#'#;#Ji:":l##.sl1'g*8,*. ir.crirt.rn Fccc& ,rn tr I lo {F L L';Hl1it5:Ll1jho5frHA$ 3[.T.XSiS]f:"ffi ttrc el&isril pn$id b ciolcLd tadr hclF. pn F. Ll;l##il::f,1H" j'"$lHsffi'Pff.pjf g:tsilrd"*l cotia&rf thc ptlwipt Ph{Goid it ccan'tcLd talh lld9cr ti{l' G.lcl xith llorl'ltl2 bgl aurid ctiaio o' ar'kt|.d r!'d io l. .b. .c.dh.lF l*f 't: lr.c D.mo.cft !l6.itf bpl llrir poai.n o{ d!. ,*Z fcE p@ri"6 ="|;f;fi.iio;'liriid^'lrra G6l* iticiio ol reombiatrt pliF*dl X" ilG-l6Cii;;d;tra-uprrim lrm orc recz acoc FDilt htbl F "r qgGricr rdr thp.trl*lsiab* |.il P.ocrl. rC$ 65?-7!9 Inl MoldgL clonl4.dt fiaf.d b lt ead ft fn pmrcr| Oleer s thc F,ldinl- t qm bcldl. A.rlcl0c llgtglftf bpl enpacllliocbryrr asie l nCl lor "'ib'dk sliaioo ol5c ah.|dra.t Fm. d..- aa.|.: ft eeFr sed b dcifnred |fr. lr, r|t .d .od 6' ld trild b dGiaru.d lt Tl rnr.L catilr.a trur Et|rrl 3 rt82. s. e.lii'ffiif#ff&re r' r. ^^tt^ Rfiltfff^.^nn .' .Atilt fistl But rl Srlt Ftl tcilY lll|rllll ^^TTCG^TAIC^AGCTTATCGA 6CT^I^GTTCCMTA6CT 3' cr^Tffclcfrcc^ccT s' Modified, Strapgene Catalog, p.347, '1999. s' .e.rcrsfofittt'iffte^rc t' a- +l T? 9m-tcr l' cGGc^TilcicTc^GtAT^arG 5 3'rcif56696c$Af ^rG 5' mFr fil,r Il rel S.I I IDI I I h 6GT ccA I t5, @ Ortl o.I pBfuescripl SK (+l-l 2958 bp rc6Accrc I 7q !'(", 5' r.,
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Subcloning: Introduction (continued) Once a gene or cDNA has been isolated, the gene is often 'mapped'. This is a procedure of digesting the DNA with a vari fy of restriction enzymes, running the ftagments on an agarose gel and determining the size ofthe fiagments. Often you need to 'subclone' the DNA into snraller fragments. One reason for this is to sequence the DNA Sequence reactions can routinely s {1uence 400 to 80O bases per reaction In this experiment, you will srbclone two HindlJllEr;rlkl fragmen(s) from Wcia faba Oeanlvefz/ cDNA T\eveinl cDNA is present as anEcoRI insert in pBSIC Finsf yott will carry out o restriction digest of pBSKVd with the restriction enzymes ^&oRI and HinN. Two ftagments will result from this digest. One student from the pair will clone the smaller band and the other student will clone the larger band. You will isolate the relevast fragmeuts and clone the fiqgment into the pBSK vector by ligation Two new plasmid constnrsts will be produced: pBSKV2 (containing the smaller fragmem) and pBSK\B (containing the larger ftagment). To ampli$ and check if the zubcloniqg wortd you will make E coli competent cells and transform them with the plasmids. Digests elminiplasmid preps and PCR. will veri$ the presence
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This note was uploaded on 07/13/2008 for the course MCDB 104 taught by Professor Lee during the Summer '08 term at UCLA.

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Preparation of insert - Cloning Strategy , I t / l_ l r\ /...

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