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Each Lab Pair Isolates Genomic DNA

You will use common chemicals instead of lab solutions to isolate genomic DNA. Buffered aspirin as the bufferPalmolive or Wisk instead of SDSIsopropanol instead of lab alcohol (ethanol)Sterile saline as the diluentAdolph's meat tenderizer or contact lens cleaner instead of phenol or protease to

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Each Lab Pair Isolates Genomic DNA

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    1. Each Lab Pair Isolates Genomic DNA Thymus (Lorraine Schepis) Wheat Germ (Irene Wu) Spinach (Nicole Brasof) Rose (Carol Champion)

    2. You will use common chemicals instead of lab solutions to isolate genomic DNA Buffered aspirin as the buffer Palmolive or Wisk instead of SDS Isopropanol instead of lab alcohol (ethanol) Sterile saline as the diluent Adolphs meat tenderizer or contact lens cleaner instead of phenol or protease to destroy nucleases

    3. Common Steps in Different Isolation Procedures Dissolve (wheat germ) or disrupt tissue Add detergent to disrupt cell membranes and also to help degrade cell walls Add meat tenderizer or contact lens cleaner to degrade nucleases Overlay lysate with alcohol to precipitate the DNA Collect the precipitated DNA by spooling or centrifugation Dissolve DNA in saline or TE

    4. Digesting Your Genomic DNA with EcoRI Each lab pair should choose best genomic sample. Each pair will prepare three different digests of this genomic DNA sample, varying the amount of DNA, as described on p. 36. Incubate the digests until 1:30 pm. During this time, prepare an agarose gel. Also, prepare 2 samples of undigested genomic DNA as described on p. 36.

    5. Pour An Agarose Gel Set up gel device, inserting a black dam at each end of the gel bed. Seal the joints with agarose. Pour your gel as you did yesterday. Overlay gel with TAE buffer after gel solidifies, as time allows. Pull comb as you did yesterday.

    6. Prepare Samples From Your Digests Prepare 3 tubes by adding 15 l TE and 2 l dye and labeling tubes 1, 2, and 3. After the incubation of your 3 genomic digests ends, put digests at 65o C for 5 min. (Why?) Cool digests in ice. PREPARE A SAMPLE FROM EACH DIGEST by transferring 5 l from each digest to the prepared tube with the same number.

    7. Gel Electrophoresis of Genomic Digests Finish preparing your gel if not already done. Load 20 l of each of 3 samples (not the digests) as directed on p. 37. Begin electrophoresis as you did yesterday. Stain and photograph gels in ethidium bromide to increase sensitivity. Follow directions in manual.

    8. Preparation of Bacterial Strains One person of each pair should streak one LB-Amp plate with JM101/pUC19 and another LB-Amp plate with JM101/pRAS2 as described on p. 38. This partner will use these plates as the source of bacteria for their plasmid DNA isolation tomorrow.

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