Manual: AdEasy Adenoviral Vector System - UCLA Human Genetics

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APPENDIX: PLAQUE ASSAY USING AGAROSE OVERLAY Preparing Viral Stock Dilutions The following protocol may be used to determine the titer (pfu/ml) of a viral stock. In addition, although the AdEasy system essentially obviates the need to plaque purify clones for a viral stock, if desired, the following protocol can also be used to isolate a single virus clone. 1. Plate AD-293 cells at a density of 5 × 10 5 per well of 6-well tissue culture plates. 2. Incubate overnight at 37°C. 3. Dilute viral stocks in 1-ml volumes over a 10-fold series from 10 –5 to 10 –9 in growth medium. Carry dilutions in duplicate. 4. Add 1 ml of each dilution to a separate well of the 6-well plate. Leave one well “medium only” (no virus added) as a control. 5. Incubate at 37°C for 2 hours. Gentle rocking during the incubation is beneficial but not required. 6. Proceed to Overlaying the Infected Cells with Agarose. Overlaying the Infected Cells with Agarose Note The agarose overlay should be applied so that it spreads fast enough to cover the plate before solidifying but not so fast that the cells are disrupted. It may be necessary to practice the overlaying technique on uninfected cells prior to performing the plaque assay. Prior to the addition of the agarose overlay, inspect the plates containing the cells to ensure adequate adherence. 1. Prepare a solution of 5% SeaPlaque ® agarose in sterile PBS, autoclave, and store in 10-ml aliquots at 4°C in 50-ml sterile conical tubes. 2. Prior to use, melt the agarose by placing the tube in a beaker of boiling water. Do not microwave the tube of agarose. 3. Once melted, cool the agarose to 45°C. 4. Add 30 ml of growth medium previously equilibrated to 37°C and mix. This makes the final agarose concentration 1.25%. Proceed immediately to the next step. 5. Completely remove the growth medium from the wells that will receive the overlay. 6. Gently pipet 3 ml of agarose/growth medium mix very gently along the side of the well and allow it to completely cover the bottom of the well. 34 AdEasy Adenoviral Vector System
7. Incubate the plate at 37°C. Plaques, having the appearance of small white spots, should be visible to the naked eye within 12–21 days. During that time, if the agarose/growth medium overlay becomes yellow, pour additional overlays at a volume of 1.5 ml per addition. 8. To determine titer, count plaques from wells where isolated plaques are clearly visible and countable. Average the counts from duplicate wells and multiply that number by the dilution factor to estimate pfu/ml. Plaque Isolation 1. Under sterile conditions, core-out well isolated plaques (as agarose plugs) with a sterile Pasteur pipette and transfer to 250 μl of growth medium in a sterile microcentrifuge tube. 2. Elute virus for 24 hours at 37°C. 3. Use this virus stock to perform an additional round of plaque purification if desired, or amplify for later use. AdEasy Adenoviral Vector System 35
Page 1 and 2: AdEasy Adenoviral Vector System Ins
Page 3 and 4: AdEasy Adenoviral Vector System CON
Page 5 and 6: AdEasy Adenoviral Vector System MAT
Page 7 and 8: NOTICES TO PURCHASER Limited Licens
Page 9 and 10: Non-Commercial Research Use License
Page 11 and 12: INTRODUCTION Recombinant adenovirus
Page 13 and 14: Pac I Kan Pac I LITR Encapsidation
Page 15 and 16: pAdEasy-1 Vector Map left arm homol
Page 17 and 18: pShuttle-CMV Vector Map kanamycin P
Page 19 and 20: pShuttle-IRES-hrGFP-2 Vector Map ka
Page 21 and 22: Recommended Primer Sequences Recomm
Page 23 and 24: Cloning Capacity of the Shuttle Vec
Page 25 and 26: Cotransforming the BJ5183 Cells to
Page 27 and 28: Interpretation of Results 5. Prepar
Page 29 and 30: XL10-Gold Transformation Protocol N
Page 31 and 32: PREPARATION OF PRIMARY ADENOVIRUS S
Page 33 and 34: Adding the DNA Suspension to the Ce
Page 35 and 36: GUIDELINES FOR INFECTION CONDITIONS
Page 37: Fluorescing cells growing in tissue
Page 41 and 42: PREPARATION OF MEDIA AND REAGENTS L
Page 43: REFERENCES 1. Benihoud, K., Yeh, P.

Manual: AdEasy Adenoviral Vector System - UCLA Human Genetics

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