274
Minimal SD base (Clontech), 765 mg CSM dropout Uracil
(CSM-Ura; Formedium), 100 mg Adenine hemisulfate, 7.4 g
KCl and dissolve in water as described in the previous step.
Mix and adjust pH to 5.5 with 1 M KOH. Make up to 1 L
with water. To obtain solid medium, add 20 g Agar (Plant cell
culture tested, Sigma-Aldrich). Autoclave and store liquid
medium at RT, pour agar plates as previously described, and
store at +4 °C.
3. Selective medium without Uracil and Methionine, supplemented with Adenine and KCl (SEL-U-M + KCl): weigh
725 mg L-Arginine, 100 mg Adenine hemisulfate, 1.5 g CSM
dropout Uracil and Methionine (CSM-Ura-Met; Formedium),
10 g D-Glucose and add to 800 mL of water. Add 2 mL of
500 mM MgSO 4 and 200 μL of 500 mM CaCl 2 . Supplement
with KCl at the desired final concentration diluting a 2 M stock
solution (see Note 3). Mix and adjust pH to 6.0 with Phosphoric
acid. Make up to 1 L with water. Autoclave and store liquid
medium at RT. Before use, add 1 μL Vitamin solution and
1 μL Trace Elements per each mL of media. For solid medium,
add 15 g Low Potassium Agar (Sigma-Aldrich #P9338). After
sterilization, allow the medium to cool down and add Trace
Elements and Vitamins as previously described; pour medium
into petri dishes. Seal solid plates and store at +4 °C in the dark
(see Note 4).
4. Vitamin solution: weigh and add in a 50 mL tube 10 mg
Biotin, 20 mg D-Panthotene acid calcium salt, 20 mg Nicotinic
acid, 20 mg Pyridoxine HCl, 20 mg Thiamine, and 10 mg
Myo- inositol. Add ultrapure grade water up to 50 mL, mix
and filter the solution through 0.2 μm syringe filter. Store at
+4 °C, in the dark (see Note 5).
5. Trace elements: weigh 25 mg Boric acid, 2 mg CuSO4, 5 mg
KI, 25 mg FeCl3, 20 mg MnSO4, 45 mg Molybdic acid,
20 mg ZnSO 4 , 500 μL HCl 37% and dissolve in 50 mL of
water. Sterilize the solution as described in the previous step.
Store at +4 °C, in the dark (see Note 5).
1. High-fidelity PCR Amplification System (e.g., Pfu DNA
polymerase).
2. QuikChange II XL Site-Directed Mutagenesis Kit.
3. GeneMorph II Random Mutagenesis Kit.
4. Restriction Enzymes.
5. Commercial PCR purification kit (e.g., QIAquik PCR
Purification Kit).
6. Commercial DNA gel extraction kit (e.g., Wizard SV Gel and
PCR Purification Kit).
2.6 Mutation
Strategies
2.6.1 Enzymes
and Reagents
Cristian Cosentino et al.
Minimal SD base (Clontech), 765 mg CSM dropout Uracil
(CSM-Ura; Formedium), 100 mg Adenine hemisulfate, 7.4 g
KCl and dissolve in water as described in the previous step.
Mix and adjust pH to 5.5 with 1 M KOH. Make up to 1 L
with water. To obtain solid medium, add 20 g Agar (Plant cell
culture tested, Sigma-Aldrich). Autoclave and store liquid
medium at RT, pour agar plates as previously described, and
store at +4 °C.
3. Selective medium without Uracil and Methionine, supplemented with Adenine and KCl (SEL-U-M + KCl): weigh
725 mg L-Arginine, 100 mg Adenine hemisulfate, 1.5 g CSM
dropout Uracil and Methionine (CSM-Ura-Met; Formedium),
10 g D-Glucose and add to 800 mL of water. Add 2 mL of
500 mM MgSO 4 and 200 μL of 500 mM CaCl 2 . Supplement
with KCl at the desired final concentration diluting a 2 M stock
solution (see Note 3). Mix and adjust pH to 6.0 with Phosphoric
acid. Make up to 1 L with water. Autoclave and store liquid
medium at RT. Before use, add 1 μL Vitamin solution and
1 μL Trace Elements per each mL of media. For solid medium,
add 15 g Low Potassium Agar (Sigma-Aldrich #P9338). After
sterilization, allow the medium to cool down and add Trace
Elements and Vitamins as previously described; pour medium
into petri dishes. Seal solid plates and store at +4 °C in the dark
(see Note 4).
4. Vitamin solution: weigh and add in a 50 mL tube 10 mg
Biotin, 20 mg D-Panthotene acid calcium salt, 20 mg Nicotinic
acid, 20 mg Pyridoxine HCl, 20 mg Thiamine, and 10 mg
Myo- inositol. Add ultrapure grade water up to 50 mL, mix
and filter the solution through 0.2 μm syringe filter. Store at
+4 °C, in the dark (see Note 5).
5. Trace elements: weigh 25 mg Boric acid, 2 mg CuSO4, 5 mg
KI, 25 mg FeCl3, 20 mg MnSO4, 45 mg Molybdic acid,
20 mg ZnSO 4 , 500 μL HCl 37% and dissolve in 50 mL of
water. Sterilize the solution as described in the previous step.
Store at +4 °C, in the dark (see Note 5).
1. High-fidelity PCR Amplification System (e.g., Pfu DNA
polymerase).
2. QuikChange II XL Site-Directed Mutagenesis Kit.
3. GeneMorph II Random Mutagenesis Kit.
4. Restriction Enzymes.
5. Commercial PCR purification kit (e.g., QIAquik PCR
Purification Kit).
6. Commercial DNA gel extraction kit (e.g., Wizard SV Gel and
PCR Purification Kit).
2.6 Mutation
Strategies
2.6.1 Enzymes
and Reagents
Cristian Cosentino et al.