BIOL 205 Lecture Notes - Lecture 15: Auxotrophy, Cloning, Calcium Chloride

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Ecori fragments if your gene has blunt ends: you"(cid:396)e i(cid:374)te(cid:396)ested i(cid:374) a spe(cid:272)ifi(cid:272) (cid:396)egio(cid:374) o(cid:374) the. Dna for a specific gene and there are no. It does(cid:374)"t (cid:272)ut (cid:448)e(cid:396)(cid:455) (cid:449)ell if (cid:455)ou just put gaattc (cid:396)ight at the 5" e(cid:374)d: can use those primers with the engineered. Ecori site and pcr it: afte(cid:396) se(cid:448)e(cid:396)al (cid:396)ou(cid:374)ds of pcr, (cid:455)ou"ll have a product now that has the. In the picture is a colour coded donor dna and let"s just sa(cid:455) that ea(cid:272)h a(cid:396)(cid:396)o(cid:449) is a (cid:396)est(cid:396)i(cid:272)tio(cid:374) enzyme site for ecori so you can essentially get a green piece, purple piece, blue piece etc. In the polylinker, those are unique sites: origin of replication (ori) If (cid:455)ou do(cid:374)"t ha(cid:448)e this, the (cid:373)a(cid:272)hi(cid:374)e(cid:396)(cid:455) (cid:449)o(cid:374)"t (cid:396)epli(cid:272)ate (cid:455)ou(cid:396) plas(cid:373)id fo(cid:396) (cid:455)ou: antibiotic selection (e. g. ampr) If you disrupt the lacz reading frame because you inserted a cloned piece in there, it does(cid:374)"t (cid:373)ake a p(cid:396)ope(cid:396) la(cid:272)) e(cid:374)z(cid:455)(cid:373)e, so the colony will be white.

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