1 / 12

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.

sydnee
Télécharger la présentation

Confirmation of pARA–R Restriction Digest

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related