AAV Titration Protocol

Fig 1. AAVpro® Titration Kit (for Real-Time PCR) Ver.2

 

1. Treat the AAV particle solution with DNase I and incubate at 37℃ for at least 15 min

(To digest free genomic DNA and plasmid DNA derived from host cells)

 

           AAV Particle Solution              2 μl

           10X DNase I Buffer                  2 μl

           DNase I                                     1 μl

           DW                                         15 μl

           Total                                       20 μl

 

2. Inactivate DNase I by heat treatment at 95℃ for 10 min.

3. Add an equal amount of Lysis Buffer (20 μl).

4. Incubate at 70℃ for 10 min.

5. Dilute the AAV vector genome solution at least 50-fold using EASY Dilution (for real-time PCR) and use directly as the template for real-time PCR.

 

 

Ⅱ. RT-qPCR

Use 5 μl of the AAV vector genome solution prepared in Ⅰ. as the template for real-time PCR. At the same time, use the Positive Control to prepare the standard curve.

 

1. Sample Preparation for Standard Curve

Dilute the Positive Control using RNase-free DW to obtain the samples for standard curve preparation. (Use 5 μl of each solution as a template for real-time PCR.)

   (1)       2 x 10^7 copies/μl (Positive Control solution)

   (2)       2 x 10^6 copies/μl (5 μl of Positive Control solution + 45 μl of DW)

   (3)       2 x 10^5 copies/μl (5 μl of (2) + 45 μl of DW)

   (4)       2 x 10^4 copies/μl (5 μl of (3) + 45 μl of DW)

   (5)       2 x 10^3 copies/μl (5 μl of (4) + 45 μl of DW)

   (6)       2 x 10^2 copies/μl (5 μl of (5) + 45 μl of DW)

 

2. Preparation of PCR mixture

 

TOPreal qPCR 2× PreMIX (SYBR Green with low ROX)    10 μl

Template DNA                                                                      1 μl

Fwd Primers 10 pmol                                                           1 μl

Rev Primers 10 pmol                                                             1 μl

Sterile water (RNase free) up to                                         20 μl

*Assemble the reaction mixture on ice

 

3. PCR Cycle

Initial Denaturation                 95℃           10~15 min

Denaturation                           95℃           10 sec

Annealing                                60℃           30 sec 

Elongation                               72℃           15~30 sec

Number of cycles         35~55 times