BCHM 4116 1st Edition Lecture 11 Outline of Last Lecture I Library Screening II Vectors and molecular cloning III PCR Outline of Current Lecture I Optimizing PCR conditions II Other PCR III Limitations IV RT PCR Current Lecture I Optimizing PCR conditions a Design a good set of primers b Choose optimal annealing temp c Mg2 concentration purity of DNA II Other polymerases for PCR a Pfu DNA polymerase has 3 to 5 exo and higher fidelity III Limitations a Contamination b Taq polymerase is error prone c Works well for shorter sequences d Specialized enzyme mix to obtain large fragments IV RT PCR to amplify a sequence from RNA V Need 2 strand cDNA a If you already have 1 strand can you start on cloning Or do you have to wait for the 2nd VI Anchored RT PCR a Rabid amplification of cDNA ends RACE b 3 RACE i forward gene specific primer anchor specific primer VII Reverse transcriptase prevents from exponential transcription VIII Cloning of PCR products IX Getting DNA into vector a RE Liagation b Recombination These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute i Clone PCR product put into vector c Gibson Assembly i Required no specific sequences but requires overlap ii Specific sequences required by recombination iii Overlap w specific vector
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