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Confirmation of pARA–R Restriction Digest

Confirmation of pARA–R Restriction Digest. Laboratory 4a. Overview. Purpose: Examine the restriction fragments that result from the double digestion of pARA – R By Bam H I and Hind III Perform gel electrophoresis to visualize DNA plasmids. Introduction.

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Confirmation of pARA–R Restriction Digest

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  1. Confirmation of pARA–R Restriction Digest Laboratory 4a

  2. Overview • Purpose: • Examine the restriction fragments that result from the double digestion of pARA – R • By BamH I and Hind III • Perform gel electrophoresis to visualize DNA plasmids

  3. Introduction • DNA fragments move at different speeds • Smaller = faster • Larger = slower • Take digested and undigested plasmid samples • Undigested = control (A-) • 2-3 bands may appear • Plasmids isolated form cells may exist in several forms:

  4. Introduction • Nicked-circle • Break in sugar-phosphate backbone • Moves slower than supercoiled • Multimer • Replicated plasmids that remain linked together • Move the slowest • Supercoiled • Compact molecule • Move quickly through gel

  5. Different Structural Forms circle “multimer” Nicked Circle Linear Supercoiled “nicked-circle” Different structural forms produce different bands.

  6. Materials • Reagents • Plasmid samples • A- • A+ • 0.8% agarose gel • 5 x loading dye • 1 x SB • DNA size marker (25 ng/μL) • Equipment & Supplies • P-20 micropipette and tips • 1.5 mL microfuge tubes • Electrophoresis equipment • Power supply • Plastic microfuge tube rack • Markers • Trans-illuminator • Camera set-up • Tube floats • Staining trays

  7. What will you need to do? • Agarose gel • Aliquot: • 10 μL of 1 Kb ladder (marker) for each group

  8. Methods

  9. Methods • 3 tubes • A- • A+ • DNA marker • Add 4 μL loading dye to tubes A- and A+ • Loading dye increases density so DNA will sink • Pump pipette to mix samples • New tip for each plasmid • Prepare the gel • Load samples

  10. Methods • Loading samples: • Use clean tip and pipette 20 μL of “DNA size marker” • Dispense as directed in Lab 1 • Using clean tip load 24 μL A- into adjacent well • Using clean tip load 24 μL A+ into adjacent well • Connect leads and turn on • As directed in Lab 1 • Let run about 30-40 minutes

  11. Conclusions • pARA–R • Should see 2 – 3 DNA bands • 3 bands in A- • 2 bands in A+ • Has enzymes

  12. 10000 8000 5000 4000 3000 2000 1500 1000 500 Restriction analysis of pARA-R Bruce Wallace Prediction for restriction gel A- A+ M A- A+ M

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