AAV Transduction Kit

Produktnummer ABIN2344790

Produktdetails

Applikation: Transfected Cell Culture (TCC)

Marke: ViraDuctin™

Proben: Cell Extracts, Cell Samples

Produktmerkmale: AAV Helper-Free System. AAV can infect both dividing and non-dividing cells and can be maintained in the human host cell, creating the potential for genome integration. Because AAV is a naturally defective virus, requiring provision of several factors in trans for productive infection, it is considered the safest viral vector to use. These factors make AAV an attractive vector for gene therapy. The AAV transduction process includes viral binding and entry, intracellular trafficking, nuclear transport, and viral second strand DNA synthesis. The viral second strand DNA synthesis has been shown to be the rate limiting step, which leads to inefficient transduction by AAV vectors. ViraDuctin™ AAV Transduction Kit is a proprietary formulation for the transduction of AAV. ViraDuctin™ provides the following advantages:
- Higher transduction efficiency in many cell types
- Easy to use
- Ideal for transduction of nonpermissive cells such as primary cells and stem cells

Bestandteile:

1. ViraDuctin™ AAV Transduction Reagent A (20X) : One sterile bottle - 5 mL.
2. ViraDuctin™ AAV Transduction Reagent B (100X) : One sterile tube - 1 mL.

Benötigtes Material:

1. AAV Stock Solution
2. Cells and cell culture growth medium 2
3. 37 °C Tissue Culture Incubator

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Increase AAV transduction efficiency in a variety of cell types
• Simple 10 minute protocol prior to host cell infection
• Ideal for use on nonpermissive cells, such as primary cells and stem cells

Protokoll: The following transduction protocol is written for adherent cells in a 6-well plate or 35 mm culture dish. Refer to the below table for the appropriate dispensing volumes of other plate formats. Culture Dish 96-well 24-well 12-well 6-well or 60-mm 10-cm 35 mm ViraDuctin™ AAV 5 25 50 100 250 500 Reagent A (20X) (μL) ViraDuctin™ AAV 1 5 10 20 50 100 Reagent B (100X) (μL) Complete Culture 94 470 940 1880 4700 9400 Media (μL) Final Reagent Mixture 100 500 1000 2000 5000 10000 (μL) Table 1: Dispensing Volumes of Different Plate Formats

1. 5The day before transduction, trypsinize and count the cells, plating 1-4 x 10 cells in 2.0-3.0 mL complete culture medium per well of a 6-well plate. Incubate cells at 37 °C overnight to ensure firm attachment.
2. Warm the ViraDuctin™ AAV Transduction Reagents for at least 10 minutes at room temperature.
3. In a sterile tube, add 100 μL of ViraDuctin™ AAV Transduction Reagent A (20X) and 20 μL of ViraDuctin™ AAV Transduction Reagent B (100X), mix by inverting.
4. Incubate 5 minutes at room temperature.
5. Next, add 1.88 mL of complete culture medium to the ViraDuctin™ Reagent mixture, mix by inverting.
6. Incubate 5 minutes at room temperature.
7. Remove the culture medium from the overnight plated cells. Apply the entire volume of ViraDuctin™ Transduction Reagent mixture to the cells (~ 2 mL).
8. Incubate at 37 °C overnight.
9. Carefully, aspirate the media from the cells.
10. Wash the cells twice with 2 mL of complete culture medium.
11. Add 1 mL of desired AAV-containing media to the plate well. Incubate at 37 °C for 1-2 hours, gently swirling/mixing every 30 minutes. 3
12. Add an additional 1 mL of pre-warmed complete media per well, incubating at 37 °C for 2-7 days.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Vorsichtsmaßnahmen: Remember that you will be working with samples containing infectious virus. Follow the recommended NIH guidelines for all materials containing BSL-2 organims.

Lagerung: -20 °C

Informationen zur Lagerung: Store kit components at -20°C until their expiration dates. Avoid multiple freeze/thaws by aliquoting.

Antigendetails

Hintergrund: The viral system includes vectors developed from retrovirus (RV), adenovirus (AdV), adeno- associated virus (AAV), lentivirus (LV), and herpes simplex virus (HSV). AAV belongs to the family of Parvoviridae, a group of viruses among the smallest of single-stranded and non-enveloped DNA viruses. There are eight different AAV serotypes reported to date. Recombinant AAV-2 is the most common serotype used in gene delivery, and can be produced at high titers with a helper virus or