BioRad Cloning Protocol

Using BioRad Biotechnology Explorer™ Microbial Culturing Kit and Ligation and Transformation Module

The following protocol will be sufficient to prepare approximately 22 samples:
20 PCR samples, one positive control, and one negative control

Preparation
Prior to beginning the protocol prepare all necessary reagents and components:

P1. Purify all PCR products using desired method of PCR purification.
At least two days prior to Ligation and Transformation:
P2. Prepare LB agar plates (4) and LB amp IPTG agar plates (1 plate/sample) with final concentrations of 50 μg/ml ampicillin and 0.2 mM IPTG.

  • Rehydrate one of the provided ampicillin vials with 3 ml sterile water to obtain a 10 mg/ml solution. Store at -20°C until ready to use.
  • Prepare LB agar by mixing the entire packet of LB powder with 500 ml of distilled water in a 1 L Erlenmeyer flask. SWIRL to mix (DO NOT SHAKE) and then autoclave for 30 minutes on the wet cycle. If no autoclave is available, heat the solution to boiling in the microwave, repeating heating and swirling until agar is completely dissolved (no clear specks of agar in liquid). Be careful to not boil solution over.
  • Allow LB agar to cool to approximately 50°C before pouring onto 4 sterile petri dishes. (These agar plates will be used to grow an E. coli starter culture).
  • With remaining LB agar (approximately 400 ml) add 2.0 ml ampicillin and 80μl IPTG (included in Ligation and Transformation module) and swirl to mix.
  • Immediately pour this mixture onto desired number of sterile petri dishes (number of samples + control plates). These plates will be used to grow transformed samples.

P3. Prepare LB broth and LB amp broth

  • Add one provided capsule of LB to 50 ml of distilled water in a 250 ml (or larger) Erlenmeyer flask. Swirl to dissolve and autoclave for 30 minutes on the wet cycle. If no autoclave is available, the solution can be sterilized in the microwave by heating to boiling a minimum of three times. Store at 4°C until ready to use.
  • If preparing plasmid minipreps after transformation, prepare LB amp broth as well. To do this, prepare the above recipe of 50 ml of LB Broth. Allow the solution to cool to 50°C and then add 250 μl of ampicillin stock (10 mg/ml) to obtain a final concentration of 50 μg/ml. Depending on the number of clones that you plan to pick, you may want to prepare several of these solutions. Store at 4°C until ready to use.

P4. Prepare E. coli starter plate

  • Rehydrate the provided E. coli HB101 vial with 250 μl of sterile water.
  • Streak each of the 4 LB agar plates with 10 μl (if using small petri dish provided with kit) or 20 μl (if using standard size petri dish) of the rehydrated bacteria and incubate overnight at 37°C.
  • Once there are visible colonies, wrap the plates in parafilm and store at 4°C until ready to use (viable for up to 2 weeks).

As late as possible one day prior to Ligation and Transformation:
P5. Prepare starter culture:

  • Dispense 2-5 ml of LB broth into four of the provided test tubes.
  • Inoculate LB broth with a starter colony from an E. coli starter plate and incubate for a minimum of 8 hours at 37°C in a shaking incubator or water bath.

Immediately prior to beginning the Ligation reaction:
P6. Begin preparing competent cells:

  • Thaw C-growth medium. For each sample, pipet 1.5 ml C-growth medium into a provided test tube and incubate at 37°C for at least 10 minutes.
  • Pipet 150 μl of fresh starter culture (from step P5) into each of the tubes containing pre-warmed C-growth media and place in a shaking 37°C water bath or incubator for 20–40 min.

P7. Prepare agar plates:

  • For each sample, label an LB amp IPTG plate with sample name and initials. Place in incubator at 37°C.

Ligation

  1. For each sample, label a microcentrifuge tube with sample name, initials, and “ligation.”
  2. Spin down the provided tubes of 2x Reaction Buffer and proofreading polymerase.
  3. In the labeled microcentrifuge tube, set up the blunting reaction with these reagents and volumes:

2x ligation reaction buffer: 5 μl
Purified PCR product: 1 μl
Sterile water: 2.5 μl
Proofreading polymerase: 0.5 μl
Total volume: 9.0 μl

  1. Close cap, vortex and centrifuge briefly to collect the contents at bottom of tube.
  2. Incubate tube in a water or dry bath at 70°C for 5 minutes.
  3. Place tube on ice for 2 minutes.
  4. Centrifuge briefly and place tube at room temperature.
  5. Spin down the provided tubes of pJet1.2 vector and the T4 DNA ligase.
  6. Set up the ligation reaction with the following reagents and volumes:

Blunting reaction (from previous step) 9.0 μl
T4 DNA ligase 0.5 μl
pJet1.2 blunted vector 0.5 μl
Total Volume: 10.0 ul

  1. Close cap, vortex and centrifuge briefly to collect the contents at bottom of tube.
  2. Incubate tube at room temperature for 5-10 minutes and then place on ice until next step.

Preparation of Competent Cells

  1. For each sample, label a microcentrifuge tube with initials and “competent cells.”
  2. For each sample, label another microcentrifuge tube with “TF buffer.”
  3. Pipet 250 μl of transformation reagent A and 250 μl of transformation reagent B into tube labeled “TF buffer.” Vortex and immediately place on ice.
  4. Transfer competent cells from step P6 (be sure that they have grown for 20-40 minutes at 37°C) by decanting or pipetting cells into tube labeled “competent cells.” Centrifuge at high speed for 1 minute and then immediately place on ice.
  5. Visually locate the bacterial pellet at the bottom of the “competent cells” tube, and then remove and discard the supernatant, carefully avoiding the pellet. *Keep cells on ice as much as possible during this procedure*
  6. Pipet 300 μl of ice cold “TF Buffer” into each bacterial pellet and resuspend by pipetting up and down. Be sure that all cells are fully resuspended with no clumps. Avoid removing the cells from the ice for more than a few seconds at a time.
  7. Incubate the resuspended bacteria on ice for 5 minutes.
  8. Centrifuge the bacteria at high speed for 1 minute. Immediately place back on ice.
  9. Remove and discard the supernatant. Resuspend a second time in 120 μl of ice cold “TF Buffer.” Be sure that all cells are fully resuspended with no clumps. Avoid removing the cells from the ice for more than a few seconds at a time.
  10. Incubate the resuspended cells on ice for 5 minutes. These “competent cells” are now ready for transformation.

Transformation

  1. For each sample, label a microcentrifuge tube with sample name, initials and “gene TF.”
  2. Pipet 5 μl of the ligation reaction from step 11 of Ligation Protocol into the “gene TF.” Store any remaining ligation reaction at -20°C. Place “gene TF” tubes on ice.
    1. *If performing a positive control reaction, pipet only 1μlof the provided positive control
    2. plasmid into a “gene TF” tube.
    3. Pipet 50 μl of “competent cells” into each “gene TF” tube containing the 5 μl of ligation reaction. Gently pipet up and down twice to mix.
    4. Incubate “gene TF” tubes on ice for 10 minutes.
    5. Retrieve LB amp IPTG plates from incubator (may be best to do one or two at a time to maintain the elevated temperature of the plate).
    6. Pipet the entire volume (55 ul) of each “gene TF” onto the corresponding labeled plate. Use a sterile inoculation loop or spreader to gently spread the bacteria on the plate.
    7. *Do not spread for more than 10 seconds…it is vital that the plate remains warm during this step to ensure high transformation efficiency.
    8. Immediately place the plates upside down in the incubator at 37°C and incubate overnight.
    9. Analyze the results the following day or wrap in parafilm and store at 4°C until ready to select colonies.

Inoculating a Bacterial Colony for Plasmid Miniprep(Complete this step only if you plan to prepare plasmid minipreps of your samples)

  1. For each colony on a plate label a 15 ml test tube with sample name, initials, and colony number.
  2. Pipet 5 ml of LB Amp Broth from step P3 into each test tube.
  3. Using a sterile loop or sterile pipet tip, pick a single colony from the LB Amp IPTG plate and inoculate the corresponding LB Amp broth tube.
  4. Place the inoculated LB Amp broth tubes in a shaking incubator at 37°C to grow overnight.
  5. Perform a miniprep reaction using the protocol of your choice.