Stock Solutions
FM
Na2EDTA (Ethylene diaminetetra acetic acid)
FeSO47H2O (Ferrous Sulfate)
500ml
1.865g
1.390g
250ml
0.932g
0.695g
Dissolve the Na2EDTA completely in 450 (225) ml H2O before adding the FeSO47H2O.
Bring to final volume. Autoclave in a 500ml (orange cap) bottle, cool and wrap bottle
with aluminum foil to keep out the light. Store in Refrigerator.
GM
Glycine
Nicotinic Acid
Pyridoxine HCl
Thiamine HCl
500ml
20mg
50mg
100mg
100mg
250ml
10mg
25mg
50mg
50mg
No Need to Autoclave. Store in 500ml (orange cap) bottle in the refrigerator.
BA (1mg/ml)
Add 500ul KOH to 50mg BA (6-Benzylaminopurine) to dissolve the BA (prepare in
50ml centrifuge tube).
Bring final volume to 50ml. Store in Refrigerator.
NAA (0.1mg/ml)
Dissolve 5mg NAA (Napthalene Acetic acid) in 45ml DD H2O in 50ml centrifuge tube.
Bring final volume to 50ml. Store in Refrigerator. Dissolve in NaOH if nesscessary
5N HCl
207ml concentrated HCl – POUR INTO 293ml deionized water
1N HCl
42.5ml concentrated HCl – POUR INTO 457.5ml deionized water
5N NaOH
20g NaOH into 100ml deionized water. Store in PLASTIC bottle.
1M KOH
Dissolve 14.025g KOH into 250ml water. Store in plastic bottle.
Kanamycin sulfate (K75)
750mg Kanamycin into 10ml DD H2O = 75mg/ml
Use syringe & needle to pull up solution, pass through sterile filter into 1.5ml
microcentrifuge tubes (1ml / pink).
Tetracycline (tet10)
Add 2.5ml 95% ethanol to 2.5ml DD H2O. Add 50mg tetracycline. Use syringe &
needle to pull up solution, pass through sterile filter into 1.5ml microcentrifuge tubes
(1ml / amber)
100 mM Acetosyringone
Dissolve 196mg of 3’5’dimethoxy-4’-hydroxy-acetophenone in a small amount of 70%
EtOH. Bring volume to 10ml with DD H2O. Use syringe & needle to pull up solution,
pass through sterile filter into a small sterile tube. (58.8mg into 3ml)
Carbenicillin (CARB50)
Dissolve 1g of Carbenicillin in 10ml DD H2O. Use syringe & needle to pull up solution,
pass through sterile filter into 1.5ml microcentrifuge tubes (1ml / blue).
Chloramphenicol (CAP100)
Dissolve 500mg of Chloramphenicol in 10ml 95% EtOH. Use syringe & needle to pull
up solution, pass through sterile filter into 1.5 microcentrifuge tubes (1ml / orange).
Timentin (300mg/ml)
Using a syringe & needle, inject 10.3ml DD H2O into the vial. Mix well, then pull up the
solution. Pass through a sterile filter into 2ml microcentrifuge tubes (2ml / clear).
Gentamycin sulfate (G60)
Dissolve 300mg Gentamycin in 5ml DD H2O. Use syringe & needle to pull up solution,
pass through sterile filter into 1.5ml microcentrifuge tubes.
Claforan (500mg/ml)
Using a syringe & needle, inject 4ml DD H2O into the vial. Mix well, then pull up the
solution. Pass through a sterile filter into 1.5 microcentrifuge tubes (1ml/clear).
Stock Solutions for GUS Staining
GUS Stain:
0.2M Na2HPO4
DD H2O
0.5M Na2EDTA
X-gluc
2.5ml
1.25ml
1.2ml
50l
2.5mg
5ml
2.5ml
2.4ml
100l
5mg
10ml
5ml
4.8ml
200l
10mg
50ml
25ml
24ml
1ml
50mg
**dissolve X-gluc in a small amount of DMSO first.
0.2M Na2HPO4 (Sodium Phosphate)
Prepare 50ml 1M stock:
Add 7.1g Na2HPO4 to 45ml DD H2O
Dissolve & adjust pH = 7.0
Raise volume to 50ml
Prepare 0.2M Na2HPO4 from this stock:
Add 40ml Na2HPO4 to 160ml DD H2O
0.5M Na2EDTA
Add 37.22g Na2EDTA to 150ml DD H2O
Dissolve by adjusting pH = 8.0
Raise volume to 200ml
3:1 Fixer
3 parts 95% EtOH – ADD ACID TO EtOH! – 1 part glacial acetic acid
DO THIS IN THE FUME HOOD
Harvesting Seeds
For Fresh Seed:
1. Immerse seed for 10 minutes in well agitated 51C distilled water.
2. Dip seeds in a 1% solution of 8-Hydroxyquinoline sulfate (1g/L)
3. Allow seed to dry, then store in sandwich size baggies in the refrigerator.
For contaiminated Seed:
1.
2.
3.
4.
5.
Immerse seed for 10 minutes in well agitated 30% bleach solution (with tween)
Give seeds a rinse with sterile water for 8 minutes
Immerse seed for 10 minutes in well agitated 51C distilled water.
Dip seeds in a 1% solution of 8-Hydroxyquinoline sulfate (1g/L)
Allow seed to dry, then store in sandwich size baggies in the refrigerator.