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$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Nucleofection of merozoites or sporozoites
- Preparation before nucleofection of parasites
- Prepare about 107 merozoites or sporozoites in one tube. If transfecting merozoites, prepare 3-4 tubes.
- Prepare an amount of plasmid DNA or purified PCR fragment that is greater than or equal to 10 µg.
NOTE: The plasmid used in this study contains 2 genes: enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii (TgDHFR-TS).
- Prepare 25 U of restriction enzyme. If plasmids are linearized, the restriction enzyme can improve the transfection efficiency. If the plasmids are circular, omit the restriction enzyme.
- Prepare 85 µL of nucleofection buffer (Table 1): mix 20 µL of nucleofection buffer I and 1 mL of nucleofection buffer II and use a part of the solution. The volume of the total buffer is 100 µL.
- Nucleofection
- Centrifuge the sporozoite or merozoite suspension at 600 x g for 10 min. Then discard the supernatant.
- In the following order, add 85 µL of nuclear transfection buffer, 10 µg of plasmid (PCR fragment), and 25 U of restriction enzyme (usually 5 µL) into the 1.5 mL tube containing sporozoites or merozoites.
- Transfer the suspension to a nuclear transfection cup. Put the cup into a nuclear transfer groove.
- Turn on the nucleofection device by using the power button and select the transfection procedure U-033. If the nucleofection device starts in the Free Program Choice mode, exit this mode by pressing the X button.
- When the program finishes, press the X button of the nucleofection device, and the screen should display OK, indicating that the nucleofection is successful.
- Add 0.5-1 mL of Dulbecco's Modified Eagle's Medium (DMEM) to the nucleofection cup to stop the reaction and transfer the suspension to 1.5 mL tube after mixing gently.
| Buffer
|
Composition
|
| Nucleofection buffer I | ATP-disodium 2g, MgCl2-6H2O 1.2g, 10 ml water |
| Nucleofection buffer II | KH2PO4 6 g, NaHCO3 0.6 g, glucose 0.2 g, 500 ml water |
| *water: distilled water | |
Table 1: Composition of Buffers.