I m working on gene deletion in Salmonella Gallinarum by using lambda red technology . I have transformed pKD46 into Salmonella Gallinarum by electroporation. But I m stuck in transformation of PCR product .. Kindly guide me what could be the possible reasons for the failure..
For pKD46 mediated gene deletion, we use the following protocol: -
1. Subculture the cells (Grown at 30 degrees) in 10 mL LB and add 1mM arabinose for induction. Grow up to 0.6 OD again at 30 degrees.
2. Wash the cells thrice with ice-chilled water in a falcon tube.
3. Transfer the cells to a microfuge tube and spin for 5 min at 5K.
4. Resuspend the pellet in 100 microliters chilled MQ and take 50 microliters for electroporation.
5. Add 300 - 500 nanograms of your PCR DNA and give the pulse.
6. Immediately add 1 mL of warm ( kept at 37 degrees) 2YT media (You can do it outside also) and keep it at 37 degrees for 4 hours.
7. Plate the cells in selective plates.
Hope it will work.
Good Luck
Electroporation work best for transformation. Alternatively you can try heat shock method as described by Jitendra Singh's
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