Lec11_W11 - Review PCR PCR animation Cloning PCR products...

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Review PCR PCR animation Cloning PCR products Where are we? Alkaline lysis method of plasmid purification
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Uses of PCR 1. Gene cloning - use PCR to amplify specific gene(s) for cloning into vectors Can add restriction sequences to 5’ends of primers Or use As on the ends of product and T- tailed vector http://biorad.cnpg.com/lsca/videos/Scientis
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Figure 9.11 Polynucleotides synthesized by Taq polymerase often have an extra adenosine at their 3’ ends. Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.
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Figure 9.12 Using a special T-tailed vector to clone a PCR product. Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.
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Figure 9.14 A PCR primer with a restriction site present within an extension at the 5’ end. Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown. Our forward primer actually had 10 additional nucleotides at its 5’ end which contained the Sac1 restriction site Our reverse primer actually had 10 additional nucleotides at its 5’ end which contained the Eco RV restriction site
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Figure 9.13 Obtaining a PCR product with a sticky end through use of a primer whose sequence includes a restriction site. Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.
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Where are we? Set up PCR of lux AB, ran PCR product on gel, and cleaned up sample using Qiagen kit (removed dNTPs, Taq, etc.) Saved this sample to clone into plasmid next week Started overnights from blue and white colonies and then prepared plasmids from colonies using Qiagen kit Digested plasmids with Sal 1 to check insert sizes Recombinant vector will be cut into two pieces one will be pGEM and one will be insert
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