Primer

RecA

Orf-hisG

Step

Temperature

Time

Temperature

Time

Cycles

Initial denaturation

94˚C

5 min

94˚C

5 min

1

Touchdown denaturation

94˚C

30 sec

94˚C

30 sec

Touchdown annealing

67˚C to 57˚C

1 min 30 sec

53˚C to 43˚C

2 min

20

Touchdown extension

72˚C

2 min

72˚C

2 min

Regular cycle denaturation

94˚C

30 sec

94˚C

30 sec

Regular cycle annealing

58.5˚C

1 min 30 sec

47˚C

2 min

20

Regular cycle extension

72˚C

2 min

72˚C

2 min

Final extension

72˚C

5 min

72˚C

5 min

1