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Construction and Packaging Service of virus Vector

Construction and Packaging Services of AAV

Large scale AAV production

Bacterial Transformation Protocol
Transformation is a technique in molecular biology to introduce  foreign DNA into bacterial cells. The ability to take up free, extracellular genetic material is the prerequisite for bacterial competent cells to undergo transformation. Bacterial transformation can obtain the replication of sequence of interest in a recombinant plasmid so that further manipulation such as cloning, virus production, or other molecular biology techniques can be performed.

Figure 1. Key steps in the process of bacterial transformation
1. Transfer the required number of competent cells from -80 ℃ to wet ice for 5-10 minutes until thawed. Gently tap the tubes multiple times to obtain uniform suspension.
2. Add 1 ng to 50 ng of purified plasmid DNA directly to cells (if desired, control DNA can add to extra tube). Mix by gentle tapping and incubate the cells on ice for 30 minutes.
3. Heat shock cells for 45 seconds with 42° C water bath.
NoteTime will vary with application and bacterial strain and it will kill bacteria by keeping them at high temps for too long.
4. Immediately transfer the cells to ice for 2 minutes.
5. Add 500 μL LB media (without antibiotic) or SOC media to the tube and grow in 37°C shaking incubator for 1 h.
6. Pipette 10-100 µL of each transformed cell suspension onto pre-warmed LB agar plates with selection antibiotic (It depends on the antibiotic marker present in the plasmid DNA) and spread it using sterile spreader or roller beads.
NoteIf blue/white screening for recombinants is required, incorporate 40μl 2% X-gal, 8μl 20% IPTG on the plate. The whole process is required to be carried out in the ultra-clean workbench.
7. Incubate plates at 37°C Cabinet Incubator overnight until the liquid on the surface penetrates into the culture medium and the agar plate dries adequately.

Important Tips:
1. If electrocompetent cells are being used, place the electroporation chamber on ice.
2. The transformation efficiency is defined as the number of transformants generated per µg of the amount of DNA added to the cells.
Transformation efficiency is calculated using the following formula:
If you have any questions,please email us at [email protected].


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