Parasite Transfection

0. The day (or two) before transfection:

For type I (RH) transfections, infect T25 with 0.5 - 1 ml for full vacuoles.
For other strains, infect T25 with 0.25 ml 2 days before you'll need them.

1. Cut DNA if necessary, phenol-chloroform clean and isoproponal precipitate resuspend DNA vector in 100μl cytomix.
Note: It's helpful to keep a small aliquot of “semi-sterile” cytomix for resuspending your DNA.

2. Have 1 confluent T25 HFF ready per transfection.

3. Parasites - one highly infected T25 will make about 3 transfections. syringe release (27g). Pellet parasites by spinning at 5-10' @ 1000g in the clinical centrifuge.

4. Wash in 10 ml cytomix, sediment as above 5-10’ @ 1000g in centrifuge.

5. Resuspend pellet in cytomix 0.5 ml cytomix per transfection + DNA. Use 10-15 μg / transfection for most applications. Using 20+ μg will greatly increase the chance of multiple integration events.

6. Electroporate cells

cuvette			2mm
resistance: 		R2(24)
charging voltage:	1.2-1.4kV
max voltage range:	2.5kV/resist

Allow blinking to stop after initial pulse. Reset after each pulse. Repeating electroporation one time per transfection may improve efficiency.

7. Incubate @ RT°C for 15', then add contents to a T25 flask with HFF monolayer

8. Move to a 37°C, 5% CO2 incubator.

8. If selecting for HxGPRT, wait overnight then change to media + 50 μg/ml MPA and 50 μg/ml Xan.

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