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southern transfer

Research Plan. Isolate Genomic DNA. Southern Blot Analysis. Digest Genomic DNA w/ Various Restriction Enzymes. Agarose Gel Electrophoresis and Southern Transfer. Make Non-Radioactive PEPCK Probe. Hyribidize Probe to Southern Blot. Washes and Chemiluminescent Detection. Data Analysis. . . . . . . Broad Overall Objective.

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southern transfer

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    1. Southern Transfer

    3. Broad Overall Objective Is Phosphoenolpyruvate carboxykinase a single or multicopy gene in E. huxleyi

    4. Todays Laboratory Objectives To become familiar with a Southern Hybridization/Analysis a. mechanics of setting up a Southern b. What kinds of information can be gleaned To be able to evaluate a restriction digest To distinguish between a partial and complete digest of genomic DNA using agarose gel electrophoresis

    5. Theoretical Basis of Southern Definition Southern analysis is the transfer of denatured DNA form an agarose gel to a membrane on which it can be analysed using a labelled complementary DNA or RNA probe. Plan 1. agarose electrophoresis2. membrane transfer (capillary transfer)3. detection (chemiluminescent)

    6. Loading the Agarose Gel Lane 1= Lambda Hind III Marker (negative control Lane 2= Genomic DNA/Bam HI Lane 3= Genomic DNA/Eco RI Lane 4= Genomic DNA/Hind III Lane 5= Genomic DNA/PST I Lane 6= Genomic DNA/Eco RI + Pst I Lane 7= Genomic DNA/Bam HI + Hind III Lane 8= PEPCK cDNA clone (positive control)

    7. Agarose Gel Electrophoresis

    9. DNA Transfer Accomplished via Capillary Action

    10. Hybridization and Detection

    11. Next Week PCR will be employed to create non-radioactively labelled PEPCK probes Homologous probe will be generated Probes will be hybridized to genomic DNA on membrane to determine which restriction fragment(s) may harbor the PEPCK gene

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