BPS3151 Study Guide - Final Guide: Chaotropic Agent, Restriction Digest, Restriction Site

• School: University of Ottawa
• Department: Biopharmaceutical sciences
• Course: BPS3151
• Professor: Luc Poitras

Document Summary

Primer design (cid:1009)" (cid:1010) nu(cid:272)leotides, (cid:396)est(cid:396)i(cid:272)tion site, (cid:272)oding se(cid:395)uen(cid:272)e 3". Pcr mix well and add all reagents: two annealing temperatures used because first cycle only involves the coding sequence where 2nd cycle has more dna (6nts + restriction site) Ta is 5 degrees lower than tm: low ta causes random base pair formation, high ta causes lower amplification because of less hydridization. Directional ligation is where two different restriction enzymes were used to ensure insert is specifically oriented and prevents recircularization. Non-directional ligation uses one restriction enzyme and promotes recircularization: alkaline phosphates used to p(cid:396)event this (cid:271)y dephospho(cid:396)ylating the f(cid:396)ee (cid:1009)" phosphate. Correct the reading frame by adding 1 or 2 nts between the restriction site and the coding sequence. Positive ligation control tests if t4 ligase can join a digested plasmid with one restriction enzyme to make sure ligase works.