This protocol was created by the Martindale lab.

Transgenic constructs were designed using the software programs Serial Cloner and Geneious Prime 2021.2.2. Inserts were G-blocked using the Twist Bioscience clanal genes tool.

Transformation

  1. Dissolve the construct in nuclease-free water to obtain a concentration of ~70-80ng/uL.
  2. Add maximum 50ng of the construct into a frozen aliquot of E. coli (50uL in a 0.5mL tube) and leave it on ice for 30 minutes.
  3. In the meantime, take out from the 4°C the yeast base and leave it on the bench. Take also out two agar plates and warm them in the incubator at 37°C.
  4. After the 30 minutes add 70uL of yeast base to each plate, then add 10uL to one warmed plate and 15 uL to the other warmed plate of the construct+E.coli mixture. We prepare one plate with a lower volume than the other to prevent having both plates with too many colonies right next to each other, which would make it hard to pick only one distinc colony for PCR. Use a swab or other tool of choice to distribute the base+E.coli on the agar.
  5. Incubate the plates at 37°C overnight.

Colony screening

  1. The morning after take out the plates from the incubator. Take out a new agar plate from the 4°C and warm it in the incubator at 37°C (~10 minutes).
  2. Take out and label PCR tubes, it would be best to screen at least 10 colonies per each construct that was transformed.
  3. Take out the warmed agar plate.
  4. Use toothpicks to pick one distinct colony from any of the 2 overnight-incubated plates. Scrape each toothpick into one spot in the warmed agar plate (mark the petri dish containing the agar with numbered squares to know the position of each screened colony) and then inside a PCR tube. Once picked all colonies needed, put the numbered agar plate in the incubator at 37°C.
  5. Prepare a mastermix for the PCR, add 24.5uL of the mix to each PCR tube containing a colony, run the PCR. Settings may vary depending on the primers used and the lenght of the construct to be amplified.
  6. After the PCR is done, run the amplified products on a 1% agarose gel and check if the construct of the right size is present.

Liquid colony cloning

  1. Pick 1-2 colonies amongst the ones containing the construct using a pipette tip. Put the tib in a tube containing 4-5mL of LB broth + ampicillin.
  2. Incubate the tube in a shaker at 37°C overnight.

DNA purification

  1. The morning after, label the following tubes: 0.5ml (for sequencing), 1.5ml (for plasmid storage), 2ml screw cap tube (for glycerol stock), a 2ml collection tube and a spin column. Spin columns and reagents are from the GeneJET Plasmid Miniprep Kit.
  2. Add 500ul of sterile 50% glycerol to each of the 2ml screw cap tubes.
  3. Remove 500ul of culture from the tube and add it to the glycerol. Close the screw cap tube. Pour 2ml of culture into the 2ml collection tube and close the cap.
  4. Put the 2ml tubes in the centrifuge and spin for 1min at max speed at room temperature. Meanwhile, take the glycerol stocks down to the -80°C.
  5. Pour the supernatant off of your bacterial pellet (into hazardous/cloning waste), add 250ul of cold resuspension solution and vortex the pellet to release it from the bottom of the tube. Return the resuspension solution to the fridge immediately after use.
  6. Add 250ul of lysis solution, mix by inversion.
  7. Add 350ul neutralization solution, mix by inversion. Centrifuge at max for 5 minutes at room temperature.
  8. Pour the supernatant into the spin column (do not disturb the pellet) and spin at room temperature on max speed for 1 minute. Discard the flow-through.
  9. Add 500ul of Wash Buffer, spin, and discard the flow-through.
  10. Repeat step 10.
  11. Spin again (dry) for 1 minutes at room temperature.
  12. Transfer the spin column to a clean/labeled 1.5ml tube, add 50ul of elution buffer, incubate for 2 minutes and spin for 2 minutes at room temperature. Discard the spin column, and determine the concentration of plasmid DNA in your sample using the Nanodrop.
  13. For sequencing, transfer 20ul of plasmid DNA (diluted to ~100ng/ul) to a 0.5ul tube. Send for 2-direction sequencing to ensure cloning the construct of interest.