Editing AAV-Production-293T-2 (section)

=== Genomic DNA isolation â TIMING Overnight incubation and 2 h  ===

58. Remove the medium from the tissue culture dishes from Steps 57A(viii) or 57B(xii), rinse once with 1Ã PBS and aspirate. Let the dishes air-dry. One dense 6-cm ESC/iPSC dish containing approximately 1 X 10^6 cells yields ~20 ÎŒg of genomic DNA. A 10-cm MSC dish containing approximately 1 X 10^6 yields up to 20 ÎŒg of DNA. 

PAUSE POINT The dishes can be wrapped in laboratory film and frozen at â 20 °C for up to 6 months, if desired. Dishes should be thawed at room temperature on a bench before use. 

59. Add 750 ÎŒl of genomic DNA lysis buffer per dish. Incubate for 10 min with gentle, occasional swirling. 

60. Scrape cells with a rubber policeman, pour into a 2-ml microcentrifuge tube. The solution should be very viscous and mostly clear. 

61. Incubate at room temperature overnight on a rotating shaker (ten rotations per min). 

62. The next day, add 4.5 ÎŒl of RNAse A (4 mg ml â 1 stock) once the DNA is in solution. Mix samples by inverting 25 times and incubate at 37 °C for 60 min. 

63. Cool samples to room temperature. Add 250 ÎŒl of protein precipitation solution to the RNAse-treated cell lysate. 

64. Vortex to mix the cell lysate with the protein precipitation solution. 

65. Centrifuge at 17,900g (typically full speed) for 10 min at room temperature in a microcentrifuge. The precipitated proteins will form a tight pellet. 

66. Gently pour the supernatant into a fresh microcentrifuge tube. If the protein pellet is not tight, then vortex, incubate on ice for 3 min and centrifuge again at 17,900g for 5 min at room temperature in a microcentrifuge. 

67. Pour the supernatant into a fresh microcentrifuge tube containing 750 ÎŒl of 100% isopropanol, leaving behind the precipitated protein. Mix the samples by inverting gently 50 times. 

68. Centrifuge at 17,900g for 5 min at room temperature in a microcentrifuge. The DNA will be visible as a small white pellet. 

69. Add 500 ÎŒl of 70% (vol/vol) ethanol and invert the tube several times to wash the pellet. 

70. Centrifuge at 17,900g for 5 min in a microcentrifuge at room temperature. Pour off the ethanol carefully. 

71. Invert and drain the tube on a clean absorbent paper and allow to air-dry for 10 min. 

CRITICAL STEP The pellet may not be very tight; drain the ethanol gently, taking care not to disturb the pellet at the bottom. 

72. Add 100 ÎŒl of TE buffer. Hydrate the DNA by incubating at 65 °C for 1 h and overnight at room temperature. 

PAUSE POINT For long-term storage, store at â 20 °C. 

Screening for targeted clones by Southern blotting â TIMING 5 d 

73. Digest 5â10 ÎŒg of genomic DNA samples with 50 U (5 U ÎŒgâ1 of DNA) of restriction enzyme for 8 h. Half of the restriction enzyme can be added at the beginning and the other half can be added after 4 h. 

PAUSE POINT The restriction digests can be stored at â 80 °C and heated to 56 °C for 3 min before loading. 

74. Pour an 0.8% (wt/vol) agarose gel in 1Ã TAE, and load 5 ÎŒg of the digested samples after mixing with the loading dye. 

CRITICAL STEP High-molecular-weight genomic DNA is sometimes not uniformly distributed in TE, which can lead to inaccurate measurement before digestion. Thus, the digested DNA samples need to be requantitated on a fluorometer before loading onto the gel. Î»/HindIII DNA can be used as size markers. 

75. Run the gel at 120 V for 4â5 h in 1Ã TAE buffer. 

76. Perform Southern blotting as described in Steps 41â54. A representative Southern blot is shown in Figure 2.<br> 

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