Aug 28, 2024
Preparation of qPCR standards for AAV titering
- Roberta Marongiu1,2,
- Santiago Unda1,2,
- Michael G. Kaplitt1,2
- 1Department of Neurological Surgery, Weill Cornell Medical College, New York, NY 10065;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Document Citation: Roberta Marongiu, Santiago Unda, Michael G. Kaplitt 2024. Preparation of qPCR standards for AAV titering. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61jndvqe/v1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 01, 2022
Last Modified: September 23, 2024
Document Integer ID: 68024
Keywords: ASAPCRN, AAV, qPCR
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
qPCR standards for AAV tittering
document annex to protocol "AAV Purification Protocol with Iodixanol gradient"
1 ug of I kb DNA = 9.1 x 1011 molecules
Use any plasmid with known size with WPRE (so you can use WPRE primers)
Digestion 100ul reaction – do not use any enzyme with star activity and make sure to use a single restriction site enzyme.
| # of reactions | volume | |
| 1 | ||
| DNA (10ug) | x | |
| 10x CutSmart buffer | 10 | |
| XbaI | 5 | |
| H2O | Up tp 95 ul | |
| V/reaction | 100 | |
Mix digestion and incubate (37oC) for minimum 2 hours up to O/N.
PCR purification and elute in 30 ul EB.
Spec very well and perform multiple readings. Spec values (ug/ml)
Calculations:
If using our pAAV.CBA.mcherry.WPRE = 6132 bases=6.132 kb
1 ug => for 1000 bp dsDNA = 9.1x1011 molecules
1 ug => for pAAV.CBA.mcherry.WPRE =9.1x1011/6.132=1.484x1011molecules
Standard 108 = 2x107 molecules
2x107 molecules weight 0.135 ng= 2x107 molecules/1.484x1011molecules
2x107 molecules/ul = 0.135 ng /ul = 135 ng/ml
When we use 5 ulin qPCR => 0.675 ng in 5 ul @ 2x107/ul
In 1 ml TE – 135 ng
Calculate the DNA volume to dilute in 1 ml of TE based on spec’ed concentration