<p> Protocol to Identify GFP Transgenic Mouse </p><p>Method to Isolate DNA from Ear Notch: </p><p>Ear Notch Buffer (80 mL):</p><p>0.298g KCl + 70 mL Sterile H2O 533 L of 1.5M TrisCl (pH 8.0)</p><p>100L of 2M MgCl2 360 L of IGEPAL 360L of Tween 20 8.647 mL Sterile H2O</p><p>Method to Isolate DNA:</p><p>1. Take 500L of ear notch buffer and add 25L of Proteinase K. 2. Aliquot 40L of buffer with Proteinase K into each Epperdorf tube. 3. Take an ear notch sample from one ear of mouse. 4. Digest 3 to 4 hours @ 56 to 65oC. Mix occasionally. 5. Take out DNA for PCR and freeze sample immediately!!!</p><p>**** Keep this DNA on ice or frozen at all times as it is will quickly degrade at room temperature!!! ****</p><p>SD-PCR Protocol: </p><p>PCR Tube Contents Volume (Total = 50L)</p><p>Sterile H2O 32.5L 10X Taq Buffer 5L 5mM dNTPs 2L 4M Primer #1 (Fwd.) 4L 4M Primer #2 (Rev.) 4L DNA Template 2L Taq Polymerase (add at SD-PCR step 2) 0.5L</p><p>Note: Do not substitute Vent for Taq as Vent will not work!</p><p>Primer #1 (Fwd.) Sequence: 5’ TGG TGA TGT TAA TGG GCA CAA 3’ Primer #2 (Rev.) Sequence: 5’ CAG CAC GTG TCT TGT AGT TCC CG 3’ SD-PCR:</p><p>1. 94oC for 10 min. (to destroy Proteinase K) 2. 80oC for 30 sec. During this time period PAUSE PCR MACHINE and add 0.5 L Taq polymerase to each tube.</p><p>3. 3 cycles at: (94oC for 1 min., 69oC for 2 min., 72oC for 1.5 min.) 4. 3 cycles at: (94oC for 1 min., 66oC for 2 min., 72oC for 1.5 min.) 5. 3 cycles at: (94oC for 1 min., 63oC for 2 min., 72oC for 1.5 min.) 6. 3 cycles at: (94oC for 1 min., 60oC for 2 min., 72oC for 1.5 min.) 7. 3 cycles at: (94oC for 1 min., 57oC for 2 min., 72oC for 1.5 min.) 8. 13 cycles at: (94oC for 1 min., 55oC for 2 min., 72oC for 1.5 min.)</p><p>9. 72oC for 7 minutes. </p><p>10. Use a 1% agarose gel to analyze the PCR product. PCR product is ~ 300 bp. </p><p>Sample gel showing desired bands indicated (arrow):</p><p> (~ 300 bp)</p><p>Lane 1: 1Kb Plus DNA ladder; Lane 5: (-) Control; Lane 6: (+) Control; Lane 7: (+ GFP Transgenic Mouse) Control; Lanes 12 & 13: PCR product of DNA isolated from ear notches. </p>