BCHM 4116 1st Edition Lecture 11Outline of Last Lecture I. Library ScreeningII. Vectors and molecular cloningIII. PCR Outline of Current Lecture I. Optimizing PCR conditions II. Other PCRIII. LimitationsIV. RT-PCR Current LectureI. Optimizing PCR conditions a. Design a good set of primersb. Choose optimal annealing temp.c. Mg2+ concentration & purity of DNA II. Other polymerases for PCRa. Pfu DNA polymerase has 3’ to 5’ exo and higher fidelity III. Limitationsa. Contamination b. Taq polymerase is error pronec. Works well for shorter sequencesd. 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 productsIX. Getting DNA into vector:a. RE/Liagationb. 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 vectorc. Gibson Assemblyi. Required no specific sequences but requires overlapii. Specific sequences required by recombinationiii. Overlap w/ specific
View Full Document
Unlocking...