370 likes | 2.43k Vues
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
E N D
General Scheme for Southern Blot Restriction Digest Gel Electrophoresis DNA Preparation: Denaturation/Depurination Transfer to filter: Blotting Detecting DNA: Probing
Southern Blot done • Restriction Digest of DNA • Electrophoresis • Denaturation/Depurination • Blotting Step (overnight) • Detection of DNA (next lab)
Format for today’s laboratory: • 5 groups • Each group • loads and runs agarose gel • Completes depurination/denaturation steps • Sets up DNA transfer
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
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
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.
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
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
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
Analysis of Southern Blot A Non-isotopic Method of DNA Detection Revised from Lab Manual Instructions
Comparing Our Experimental System to Southern Blots in Research Labs
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.