Inverse PCR - Trevor Epp August 10, 2004 Inverse PCR...

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Trevor Epp August 10, 2004 Inverse PCR Gep-SD5/Gen-SD5 (5’ Flanking) A. Genomic DNA Isolation from 96-well Plates and Restriction Analysis 1. Aspirate media and wash twice with PBS 2. Add 40 µ l tail buffer with 0.5 mg/ml proteinase K 3. Seal with parafilm and incubate at 56 o C overnight in lunch box container 4. Add 80 µ l freshly prepared NaCl/EtOH mix to each well (15 µ l 5M NaCl per ml EtOH) 5. Let sit undisturbed for 1-2 hours at room temperature 6. Lay paper towels over the wells and slowly invert 7. Wash 3X with 70% EtOH at room temp, slowly inverting as before 8. Air dry 9. For first replicate add 50 µ l TE, wrap with parafilm and store at 4 o C 10. For second replicate add 35 µ l restriction enzyme mix to each well 385 µ l 192.5 µ l 10X buffer 4 µ l 2 µ l 100mM spermidine 55 µ l 27.5 µ l RNAse A 26 µ l 13 µ l 100U/ µ l Hin dIII 3380 µ l 1690 µ l DDW 3850 µ l 1925 µ l 11. Seal with parafilm and incubate overnight at 37 o C in humidified lunch box container 12. Run 15 µ l on a 0.8% agarose gel at 18-20V overnight to check digests B. Ligation 1. Add 100 µ l DDW to 2 µ l Hin dIII digested DNA and heat-inactivate at 65 o C for 30 minutes 2. Cool to 4 o C 3. Add 300 µ l of ligation mix: 40 µ l 10X ligation buffer (total = 1ml):
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Inverse PCR - Trevor Epp August 10, 2004 Inverse PCR...

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