Could not get 2 bands (plasmid and genomic dna) after conjugation

Can you provide the protocol you are using?

1. Inoculate 2ml LB of recipient cells O/N at 37C.

2. Inoculate 2 ml LB + antibiotic (KM30) of donor cells O/N 37C.

3. Next day, transfer 200micrl of donor strain and 200micrl of recipient strain into a 1.5ml Eppendorf tube containing 800 micrl LB. Centrifuge at 12 000g for 1 min.

4. Discard the supernatant and resuspend the pellet in 1ml LB. Repeat (4) once again and resuspend the pellet in 50micrl LB.

5. Drop the suspension onto the centre of a sterile 0.45micrM filter disc placed on top of LB agar. Place the agar in a sterile container and incubate at 30C for at least 6 hrs.

6. Add 400micrl of 0.9% NaCl / LB to the dried cell pellet and dislodge the bacteria from the filter.

7. Transfer 4 x 100micrl onto LB agar containing desired antibiotic plates and store in 37C for incubation for at least 12 hrs.   

Post Merge: 9 years ago

My PCR conditions are;

94C 3mins
94C 30s
52C 30s
72C 30s
72C 2mins 30s

Primer 1 Tm (63C)
Primer 2 Tm (57C)