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.
 * 6) *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.
 * 7) *You will need to calculate the volume of DNA stock needed to provide this amount.
 * 8) Setup a 1.7mL centrifuge tube for each sample. Use an additional tube to hold MilliQ water for the blank/control samples.
 * 9) Pipette 200uL of the IJ1126 culture into each tube.
 * 10) *Use the chilled pipette tips.
 * 11) *Work quickly because it is very important to keep the culture suspension cold in this step.
 * 12) Add the assigned amount of DNA to each centrifuge tube.
 * 13) *use the chilled pipette tips.
 * 14) *Be careful not to touch the lip of the DNA stock because it is easily contaminated
 * 15) *Be careful not to get ice on the inside of the centrifuge tube lid because the ice may fall into the tube.
 * 16) *Always add DNA to the centrifuge tubes in numerical order to avoid confusion.
 * 17) *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.
 * 18) Allow the DNA/IJ1126 mixture to incubate on ice for 30 minutes.
 * 19) During the 30 minute incubation, prepare a tube of warm soft agar for each sample:
 * 20) *Obtain soft agar from the 55*C incubator.
 * 21) *Microwave the bottle of the soft agar for 50 seconds at 50% power.
 * 22) *Place paper towels under the bottle and use paper towels when handling the bottle because the soft agar may boil.
 * 23) Pipette 3mL of soft agar into each of the small test tubes on the heat block (~42-45*C).
 * 24) *Pay special attention to sterile technique.
 * 25) Obtain the T-broth agar plates from the 37*C incubation room- they should be warmed up.
 * 26) Label one plate for each tube/sample with the number corresponding to the tube.
 * 27) After the 30 minute incubation, heat shock each sample one by one:
 * 28) *Spin down the tube briefly using the mini centrifuge.
 * 29) *Use the 1000uL micropipette to suck up the whole mixture out of the tube.
 * 30) *Eject the mixture into a hot soft agar test tube.
 * 31) **Pay special attention to sterile technique.
 * 32) *Tap the test tube on the lab bench lightly to mix for 5-15 seconds.
 * 33) *Pour the test tube onto its corresponding plate.
 * 34) *Allow the soft agar to solidify.
 * 35) When the soft agar layer has solidified, place the plates in the 37*C incubation room to incubate for 3-5 hours.
 * 36) After the 3-5 hour incubation, retrieve the plates and count the plaques.
 * 37) *To stop the plaques from growing and to keep the plates, place them in the 4*C fridge.