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

== AAV titering using neutral gel electrophoresis and Southern blotting  ==

30. To prepare plasmid standards for quantitation, digest the same vector plasmid used to prepare the stock, such that the vector genome is released from the surrounding inverted terminal repeats. <br>Set up the restriction digest by adding the following to a microcentrifuge tube: <br>restriction buffer (10Ã, 2 ÎŒl); <br>plasmid DNA (1,000 ng); <br>restriction enzyme (5 U); <br>and dH2O to a final volume of 20 ÎŒl. 

31. Incubate the restriction digest in a 37 °C water bath for 1 h. Stop the reaction by adding 2 ÎŒl of 0.5 M EDTA. 

32. Run 2 ÎŒl of the restriction digest on a 1% (wt/vol) agarose gel to confirm that the plasmid is completely digested. 

33. Accurately quantify the digested DNA content by using a fluorometer and make serial dilutions as follows: 100, 30, 10 and 3 pg/ÎŒl. 

34. Prepare 1% (wt/vol) agarose gel in 1Ã TAE. Allow it to set at room temperature for 1 h. 

35. Label four microcentrifuge tubes as 1,000, 300, 100 and 30 pg. Pipette out 10 ÎŒl from each standard dilution into the appropriate tube. 

36. Take the AAV stock out of the â 80 °C freezer and thaw at room temperature. 

37. Prepare two microcentrifuge tubes, each containing 3 ÎŒl of TE and 1 ÎŒl of 6Ã loading dye. Add 2 ÎŒl of the 1:10 vector stock dilution to the first tube and 2 ÎŒl of the 1:100 stock dilution to the other (dilution preparation described in step 19, Box 4). 

38. Clip the AAV stock tubes tightly. 

39. Boil them for 2.5 min, snap-cool quickly on ice.<br>CRITICAL STEP The vector is boiled to disrupt its capsid and to release the single-stranded DNA genome before loading it onto a neutral gel. 

40. Load the standards and AAV stock dilutions along with Î»/HindIII marker on the 1% (wt/vol) agarose gel and run at 120 V for 4 h. 

41. Stain the gel in 1Ã TAE containing 0.5 ÎŒg ml â 1 of ethidium bromide for 30 min. The gel can be destained in dH2O for 20 min if required. Photograph on a UV transilluminator with a fluorescent ruler by the side. 

42. Transfer the gel to a tray and acid-depurinate with 500 ml of 0.25 M HCl for 10 min, twice. Rock the gel gently. 

43. Remove the HCl solution and neutralize with 500 ml of 0.4 M NaOH for 15 min, twice. Rock the gel gently. 

44. Transfer to a Hybond-XL membrane with 1 liter of 0.4 M NaOH overnight using standard Southern blot wicking methods63. 

45. The next day, briefly rinse the membrane in 2Ã SSC. 

46. UV crosslink the membrane on a UV Stratalinker (auto-crosslink setting).<br>PAUSE POINT The membrane can be stored at 4 °C, wrapped in plastic wrap, for several months. 

47. Prehybridize the membrane with 14 ml of Church buffer containing 150 ÎŒg ml â 1 of salmon sperm DNA (boiled for 5 min and snap-chilled on ice) for 3â4 h in a hybridization cylinder rotating in a hybridization incubator at 65 °C. 

48. Synthesize a radiolabeled probe by random priming with a Rediprime II kit according to the manufacturerâs instructions.<br> CAUTION Follow all relevant institutional guidelines while working with radioactivity. Radioactive probe must be prepared in a fume hood. Wear protective clothing including gloves, plastic shield and safety glasses. 

49. Purify the radiolabeled probe with a ProbeQuant G-50 microcolumn. Denature it by boiling for 5 min and snap-cool on ice.<br>CRITICAL STEP Use screw cap tubes for boiling to prevent leakage of radioactive probe. 

50. Add the denatured probe to the prehybridization solution and incubate at 65 °C overnight in the hybridization oven. 

51. The following day, remove the radioactive hybridization solution and dispose of it properly. Wash the hybridized membrane twice with 45 ml of Southern wash solution 1 for 15 min at 65 °C in a hybridization cylinder. 

CAUTION Follow institutional guidelines while working with radioactivity. The radioactive waste must be discarded in the designated radioactive waste disposal container. Wear protective clothing including gloves, plastic shield and safety glasses. 

52. Wash the membrane twice with 45 ml of Southern wash solution 2 for 15 min at 65 °C in a hybridization cylinder. 

53. Expose the membrane to X-ray film at â 80 °C overnight. Develop the X-ray film on a Kodak 480 RA processor. This provides a visual record of the stock titer. 

54. Measure the radioactive signal by placing the membrane in a PhosphorImager cassette for 30â60 min and analyze on a PhosphorImager using ImageQUANT densitometry software. 

55. Generate a standard curve based on the radioactive signal of the standard dilutions and determine the vector titer. The radioactive signal from the vector sample(s) should fall within the standard curve and can be used to calculate the number of genome-containing vector particles per ml. Accurate titering of the vector stock is critical for performing infections at controlled multiplicity of infections (MOIs), which is known to influence targeting frequencies.