GF1040
GF1040-Fig1

Cloning Fast Screening Kit #GF1040

$49.00–$149.00

The Gene-Foci cloning fast screening kit is designed as an initial screen for the positive clone after ligating the target gene into a vector and E.coli transformation.

For research use only.

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SKU: GF1040. Categories: , .

Product Description

The Gene-Foci cloning fast screening kit is designed as an initial screen for the positive clone after ligating the target gene into a vector and E.coli transformation. The kit can identify positive clones by size difference without mini-prep DNA extraction from transformants.

Kit content:

2 X 500 µl Fast Screen buffer.

Features:

Initial identification of the positive clones containing the gene insert without mini-prep DNA extraction, enzyme digestion. Saves time and labor.

Just 20 µl of liquid culture or half of a medium-sized colony is sufficient for screening.

Bright-colored gel loading dye included, easy gel loading after incubation.

Storage:

Store at room temperature.

 

 Protocol:

 

Before Start:

  • Warm up a water bath or heat block to 70°C
  • Warm up the fast screen buffer to >37°C to let the precipitate get back into solution.
  1. Perform enzyme digestion, ligation, and E.coli competent cell transformation. Also transform the empty vector into E.coli as a control.
  2. Pick 10~30 transformants, set up 5 ml culture in liquid LB media plus the appropriate antibiotics, also set up a 5 ml culture for the empty vector as a control. Culture in a 37°C shaker overnight.
    Alternatively, if the colonies are >2mm in diameter, you can skip the liquid culture step, directly pick half of a colony with a sterilized tooth pick, transfer to a 1.5 ml tube, and precede to step 4.
  3. Pellet 20~100 µl overnight E.coli culture at 13,000 rpm for 30 seconds, carefully aspirate or pipette off the supernatant without disturbing the cell pellet.
  4. Resuspend the cell pellet in 20µl fast screen buffer by gently pipetting up and down, avoiding bubbles.
  5. Heat at 70°C for 10 min.
  6. Cool down to room temperature and run out the entire 20 µ on a 0.7% – 1% agarose gel.
  7. Pick the clones that migrate slower than the vector control, mini-prep to extract DNA from the original 5 ml culture,  then further validate by enzyme digestion and/or DNA sequencing.

 

Fig1. An example of selecting positive clones from an experiment attempting to clone a target gene into a very difficult to handle Lentiviral plasmid using Fast Screen buffer.

Helpful hints:

  • Make sure gel running buffer is level with the surface of the gel, and not above the level of the gel.
  • Remove running buffer from the wells of the gel using a pipette to avoid overflow when loading samples.
  • Pipette lysate up and down several times before loading onto gel to reduce viscosity.