Lab protocols for the Lowe-Power lab (scroll down for table of contents Readme)

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Colony PCR

Writing/editing credits: Tiffany Lowe-Power Updated 20210118

General Tips

Colony PCR for E. coli

This “protocol” is an outline that you should amend with manufacturer’s instructions and internet searches.

Step 1: Patch E. coli and create liquid overnights

  1. Label a patch plate (LB+antibiotics) like the photo below. You will screen multiple “clones” of your construct. We refer to these as “pTLP54.1”, “pTLP54.2”, … Date the plate. Initials are optional (if not included in the plasmid name)

Photograph of an E. coli Patch plate

  1. Prep LB+antiobiotic in a 48-well deep plates with baffles (autoclavable and reusable). In your notebook, record the planned location where you will put each colony (use a plate layout excel template). Use the AliQ pipette handler to repeatedly dispense the broth.
  2. Using 1 sterile toothpick per colony, pick a silnge well-isolated colony. Patch it over a number on the patch plate. Then swirl the toothpick in the broth and discard.
  3. Incubate the 48-deep well plate at 37C with shaking overnight. Incubate the patch plate at 37C stationary overnight.

Step 2: Colony PCR

Set up the reactions on wet ice. If appropriate for the vector (Inspecting vector seq on Benchling can help you determine this), use M13F/R primers for PCR screen. Alternatively, use a pair of your Gibson cloning primers to PCR screen.

  1. Use Benchling to plan your PCR screen approach (i.e. which primers to use) and the ‘pcr_workbook.xlsx’ to plan your PCR Mastermix using a cheap Taq-PCR kit. Plan to use the parental vector as a negative control, if you have the parental vector.
    • Use 1 ul of colony lysate as the template DNA
    • Do 10 ul reactions per colony.
  2. Using a P200 multichannel, add 90 ul (sterile) milliQ water to PCR strip tubes. (n=however many colonies you are screening). Add 10 ul of the overnight liquid culture to the PCR strip tube. In the thermal cycler, incubate at 96-98C for 10 minutes.
  3. Prepare the PCR mastermix, and aliquot to PCR strip-tubes.
  4. Use P20 multichannel to add 1 ul colony lysate to PCR mix.
  5. Run a PCR reaction on the thermocycler with a program that matches the PCR kit, necessary extension time, and an anneal temperature suitable with your PCR screening primers.

Step 3: Run Agarose Gel to determine result of PCR screen

Cleaning up note: 1x LIBOR tends to grow mold in our lab. Don’t leave it in the gel boxes. Pour it into a 1L bottle. Cap. Reuse until it’s moldy, then start a new batch.

Colony PCR on Ralstonia colonies

Ralstonia has always proven tricky for colony PCR. I suspect this is due to the high GC genome (difficult PCR targets) and something inhibitory to the PCR (maybe EPS?).

Only colony PCR after selected colonies have been re-struck for purity and grown.

Validate the primers & PCR kit

This can be performed simultaneously with screening putants (putative mutants) or while you are waiting for putants to appear.

Step 1: Prepare cell lysate / boil prep

  1. Grow overnight culture in CPG (as single tubes or in 24 well plate).

  2. Transfer 50 ul culture to PCR strip tubes. Note: Consider adding glycerol to the rest of the culture (20% final vol) and saving the plate as a temporary freezer stock while you work to screen the strains. This way, you don’t need to worry about the strains accumulating spurious mutations on the plate.

  3. Add caps to PCR strip tube and run a “lyse” thermocycler program (10 min at >96 C; hold at 22 C).

  4. Transfer tubes to -20 C until frozen.

  5. Thaw. Spin for ~1 min in PCR tube centrifuge. XXX Timing might need to be updated here. Not sure if a PCR tube centrifuge will pellet cell gunk or whether this should be skipped. If pelleting is unsuccessful, maybe rewrite this steps as “add 200 ul water with multichannel to dilute PCR inhibitors in cloudy lysate”

Step 2: Run PCR

  1. Plan your PCR screen approach as described in the section below (i.e. which primers to use) and the ‘pcr_workbook.xlsx’ to plan your PCR Mastermix using a PCR kit that is effective with multi-kb GC-rich template DNA (e.g. Phusion).

  2. Run with controls:
    • “no template” negative controls
    • Parental genotype negative control
    • Positive control if possible (not possible in many cases, but is possible if screening for miniTn7 integration into the genome).
  3. If you identify the correct genotype, save the strain. See: Glycerol Stocks protocol

Problem solving

xxx to do: update this

Primer Design: Detecting an unmarked deletion

Schematic showing where to place primers for PCR screening knockout mutants

Things to update on this protocol: