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MolCellEng_Notes2 - 1 Making a genomic library 1 Have...

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1. Making a genomic library 1. Have genomic DNA & plasmid (with origin of replication-so can self-replicate and selectable marker gene-drug resistant gene) with restriction sites 2. Cut restriction sites with restriction enzyme 3. Anneal and ligate 4. Get: vector only ring, DNA only ring, VECTOR + DNA ring. 5. Transform to E. coli 6. Select for (DRUG) resistance --- so only the vectors containing the resistant drug gene survive (vector alone and vector + DNA survive) 2. E. Coli Transformation 1. Electroporation 1. Electrical shock = rotation of lipid molecules = pores in membrane that vectors can enter 2. Chemical Transformation 1. Treat cells with divalent ions such as CaCl 2 , add DNA (Vector), and brief heat shock. – DNA introduced into the cells 3. Making a cDNA library 1. Step 1: Make cDNA library 1. Isolate RNA & purify from cell; isolate away from ribosome and tRNAs 2. Bind polyA tail of mRNA onto polyT tail of oligo dT 2. Step 2: Obtain cDNA from RNA 1
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1. mRNA treated with enzyme reverse transcriptase (get the DNA code you started with originally) 2. another enzyme then make second strand of DNA – cDNA (complementary DNA) [complementary DNA is double stranded, contains original DNA template seq. used to make the mRNA and its complement) 3.
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