User:Kalkao/notebook/Transfection of T7 DNA

This protocol was developed from the supplementary information of the "Refactoring bacteriophage T7" paper.

1. Place all of the pipette tip boxes in the -20*C fridge to cool before use.
2. Place a test tube in the heat block for each transfection sample.
3. Place T-broth agar plates in the 37*C incubation room to warm up the plates- one for each sample.
4. Refill the ice bucket with the IJ1126 cell suspension to keep the cells cold.
5. Obtain the phage DNA stock, and note the concentration.
   • The optimal amount of DNA to use is 1e9 to 4e9 molecules for the conditions used in the Preparing Competent Cells for Phage Transfection protocol.
   • You will need to calculate the volume of DNA stock needed to provide this amount.
6. Setup a 1.7mL centrifuge tube for each sample. Use an additional tube to hold MilliQ water for the blank/control samples.
7. Pipette 200uL of the IJ1126 culture into each tube.
   • Use the chilled pipette tips.
   • Work quickly because it is very important to keep the culture suspension cold in this step.
8. Add the assigned amount of DNA to each centrifuge tube.
   • use the chilled pipette tips.
   • Be careful not to touch the lip of the DNA stock because it is easily contaminated
   • Be careful not to get ice on the inside of the centrifuge tube lid because the ice may fall into the tube.
   • Always add DNA to the centrifuge tubes in numerical order to avoid confusion.
   • Spin down the tube quickly before adding the DNA so the culture is at the bottom of the tube and will not leave the tube when it is opened.
9. Allow the DNA/IJ1126 mixture to incubate on ice for 30 minutes.
10. During the 30 minute incubation, prepare a tube of warm soft agar for each sample:
    • Obtain soft agar from the 55*C incubator.
    • Microwave the bottle of the soft agar for 50 seconds at 50% power.
    • Place paper towels under the bottle and use paper towels when handling the bottle because the soft agar may boil.
11. Pipette 3mL of soft agar into each of the small test tubes on the heat block (~42-45*C).
    • Pay special attention to sterile technique.
12. Obtain the T-broth agar plates from the 37*C incubation room- they should be warmed up.
13. Label one plate for each tube/sample with the number corresponding to the tube.
14. After the 30 minute incubation, heat shock each sample one by one:
    • Spin down the tube briefly using the mini centrifuge.
    • Use the 1000uL micropipette to suck up the whole mixture out of the tube.
    • Eject the mixture into a hot soft agar test tube.
      • Pay special attention to sterile technique.
    • Tap the test tube on the lab bench lightly to mix for 5-15 seconds.
    • Pour the test tube onto its corresponding plate.
    • Allow the soft agar to solidify.
15. When the soft agar layer has solidified, place the plates in the 37*C incubation room to incubate for 3-5 hours.
16. After the 3-5 hour incubation, retrieve the plates and count the plaques.
    • To stop the plaques from growing and to keep the plates, place them in the 4*C fridge.