1 / 15

Southern Blotting

Southern Blotting . General Scheme for Southern Blot. Restriction Digest. Gel Electrophoresis . DNA Preparation: Denaturation/Depurination. Transfer to filter: Blotting. Detecting DNA: Probing. Southern Blot Analysis. Southern Blot. done. Restriction Digest of DNA Electrophoresis

yale
Télécharger la présentation

Southern Blotting

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. Southern Blotting

  2. General Scheme for Southern Blot Restriction Digest Gel Electrophoresis DNA Preparation: Denaturation/Depurination Transfer to filter: Blotting Detecting DNA: Probing

  3. Southern Blot Analysis

  4. Southern Blot done • Restriction Digest of DNA • Electrophoresis • Denaturation/Depurination • Blotting Step (overnight) • Detection of DNA (next lab)

  5. Format for today’s laboratory: • 5 groups • Each group • loads and runs agarose gel • Completes depurination/denaturation steps • Sets up DNA transfer

  6. DNA Fingerprinting Analysis40 ul sample per well

  7. Depurination and Denaturation Steppage 3-83 with changes • Place gel in 100 ml 0.25N HCl for a maximum of 8 minutes. • Rinse gel several times with 100 ml distilled water • Place gel in 100 ml DNA denaturation solution for 15 min. with occasional shaking. • Replace with 100 ml fresh denaturation solution for 15 minutes

  8. The reason for depurination • Depurination: 0.25 N HCl (used to help in a teaching lab) • Reduces binding between DNA strands by creating apurinic sites (loss of purine base) • Predisposes the DNA-phosphate backbone to cleavage by alkaline solution • Smaller fragments transfer better

  9. The reason for denaturation: • Denaturation:NaOH/NaCl • Double-stranded DNA is converted to Single-stranded DNA • Sugar-phosphate backbone is cleaved at the sites of depurination • This step must always be performed.

  10. Setting up the Southern Blot Transfer page 3-84 • Line a tray with plastic wrap • Place “denatured” gel upside down on wrap • Pre-wet nylon membrane in denaturation solution for 5 minutes • Place nylon on top of inverted gel

  11. Setting up the Southern Blot Transfer Page 3-84 • Place filter paper on top of nylon membrane • Remove air bubbles • Place stack of paper towels on top of filter paper • Place empty 400 ml beaker on towel • Incubate overnight

  12. Setting up the Southern Blot Transfer • Order of Components from the bottom: • Gel • Membrane • Filter paper • Paper towels • Weight • Overnight Incubation at Room Temperature Single stranded DNA will diffuse by capillary transfer from gel to nylon membrane

  13. Analysis of Southern Blot A Non-isotopic Method of DNA Detection Revised from Lab Manual Instructions

  14. Comparing Our Experimental System to Southern Blots in Research Labs

  15. Non-isotopic Detection of DNAThese directions REPLACE those found on 3-86 3-89 • Place the membrane with the DNA side up in 100 ml of Blue-Blot solution. • Soak the membrane at room temperature for 10-15 minutes. • Remove the membrane with forceps and rinse in 200 ml distilled water. • Replenish the distilled water 3-4 times or until the membrane is destained and DNA bands are clearly visible.

More Related