MBIO 3410 lecture Nov 13 2007

MBIO 3410 lecture Nov 13 2007 - Last Lecture Overview...

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1 Last Lecture Overview Genomic Libraries Mechanical shearing Restriction enzyme digestion Choice of Restriction Enzyme Linkers or adaptors Restriction enzymes that generate sticky ends Making the library Representative Gene Library cDNA Library Reverse Transcriptase Isolating mRNA Checking integrity of mRNA Cloning of cDNA Nick translation Homopolymer tailing
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2 cDNA Libraries
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4 Treatment of cDNA ends Blunt-end ligation of large fragments is not efficient Special nucleic acid linkers are added to create sticky ends for cloning 1. Prepare the cDNA for the addition of linkers via single-strand specific nuclease (S1 or mung bean nuclease) to remove protruding 3’-ends
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5 2. Treatment with Klenow fragment of DNA polymerase I and dNTPs to fill in any missing 3’ nucleotides 3. If the linkers contain a restriction enzyme site that is likely to be found in the cDNA ( EcoR I) then the DNA should be methylated ( EcoR I methylase) before the linkers are added This will ensure the restriction enzyme cannot cleave the DNA internally
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6 4. The linkers can be ligated to the blunt-ended duplex cDNA using T4 DNA ligase This step will add one linker to each end of 5’- phosphorylated cDNA if the linkers are not phosphorylated 5. Restriction enzyme digestion ( EcoR I) generates sticky ends ready for ligation to the vector
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Directional cDNA Cloning The oligo-dT primer contains additional sequences at the 5’-end that encode a Xho I restriction enzyme recognition site (5’-CTCGAG-3’) Xho I restriction enzyme recognition site will be incorporated into the 3’-end of the cDNA The second cDNA strand contains the oligo-dG sequence with an EcoR I restriction enzyme recognition site (5’-GAATTC-3) at its 5’-end
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MBIO 3410 lecture Nov 13 2007 - Last Lecture Overview...

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