PCR Method to Introduce Mutation into YanO
PCR for N-box
add the following to PCR reaction tube
2 µl 10X buffer (tag)
1 µl (2 mM DNTP’s)
1 µl (10 µM CaP primer)
13 µl (0.1nM N-Box primer)
1 µl Taq (5 units/µl)
2 µl Template DNA (3.4 ng and 340 pg)
20 µl total
Reaction 30 cycles
Denaturation 94°C 1 min
Annealing
60°C 1 min (look up Tm for your primers)
Extension
72°C 1 min
Run Reaction on 1% Agarose Gel
Cut out bands and use Qiagen II (gel extraction kit) to purify DNA
Digest DNA with Restriction Enzymes
16 µl DNA (from purification)
2 µl 10X Buffer #2 (NEB)
1 µl EcoR1
1 µl BamH1
20 µl total
Cut out bands that give correct band size and purify DNA as above
Ligate DNA with Vector
ligate bands with pCa (vector) cut with E/B
16 µl DNA (from 2nd purification)
1 µl Vector pCa E/B
2 µl 10X ligase buffer
1 µl ligase
20 µl total
place at 16° C overnight
Transform by Electroporation
Grow up Colonies
Mini-prep
Sequence
Inject DNA