PEI Calculator for Planning AAV Packaging Transfections

As the saying goes, “failing to plan is planning to fail.” Preparation is a key step to any experiment and can help prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To get these ratios right, you need to do a few calculations. This calculator can do the math for you and it’s simple to make adjustments for the type of tissue culture dish and number of dishes used. Here’s to making AAV packaging a little easier!

Click here to download the PEI calculator for AAV packaging

Use the PEI transfection calculator for AAV packaging

1. Select the plasmid molar ratio you would like to use for your AAV packaging transfection. In my lab we regularly use the ratios below:
1. 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR
2. 3 Transfer Plasmid : 5 pHelper: 2 RepCap
   
   
2. Select what type of tissue culture dish you will be performing the transfection in:
1. 15 cm dishes OR
2. CF5 stacks
   
   
3. Select how many dishes or stacks you plan to transfect:
1. 1 to 10
   
   
4. Fill in experimental details on the spreadsheet:
1. Name your viral prep
2. Enter the specific names of the plasmids you plan to use
3. Enter the base pair size of those plasmids
4. Enter plasmid concentration

The spreadsheet will calculate the following

1. μg of each plasmid needed per dish or stack. Calculations are based on using 28 μg of total plasmid (Transfer, helper, and RepCap added together) per 15 cm dish or 2500 μg of plasmids per CF5 stack.
2. Total ug of each plasmid needed for the whole experiment
3. Total volume (μL) of plasmid needed for the whole experiment

And here’s the equation the calculator uses to give you the μl needed of each plasmid. Each part of the equation is labeled with its corresponding column in the calculator spreadsheet:

We hope that this calculator will make it easier to plan your AAV packaging experiments. Let us know what you think about the calculator in the comments section below.

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References

1. Choi, Vivian W., et al. "Production of Recombinant Adeno‐Associated Viral Vectors for In Vitro and In Vivo Use." Current Protocols in Molecular Biology (2007): 16-25. PubMed PMID: 18265393.

Additional Resources on the Addgene Blog

• Important Considerations When Using AAVs
• AAVs Are Great Tools for Gene Expression in Mammals

Resources on Addgene.org

• Find AAV Packaging and Transfer Plasmids Here
• New to Making AAV? Check out Addgene’s Step-By-Step Protocol
• Pressed For Time? Order Ready-to-Use AAV from Addgene