I'm trying to knock out a gene in E. coli BW25113 ΔclpS using the phage λ Red recombinase gene knockout method. I got a few kanamycin resistance colonies (confirmed by colony PCR) but without gene deletion.

Here are the steps that I used,

1. Transform pKD46 into BW25113 ΔclpS.

2. Pick a colony (or glycerol stock) and grow the bacteria in LB broth with carbenicillin overnight at 28C.

3. Subculture: Transfer the overnight grown 50 μl of the liquid culture into the fresh 3 ml of LB broth with carbenicillin and 10 mM L-arabinose.

4. When OD is around 0.6, put on ice and spin down at 4C.

5. Wash in 10% glycerol 3 times and resuspend 100 ul of 10% glycerol.

6. Electroporate (mix with 10 ul PCR product (~100 ng/ul))

7. Add 1 ml SOC medium and recover at 37C for 2 hours.

8. Plate 100 ul and the rest on LB with kanamycin plate at 37C.

9. If no mutation, plate the rest the next day.

What should I improve on my protocol?