Appendix B: Solutions and Recipes
Solutions for the Trebuchet Plant gDNA Extraction Protocol
Extraction Buffer
100 mM Tris, pH8.0 (50 ml of 1 M)
50 mM EDTA, pH8.0 (50 ml of 0.5 M)
500 mM NaCl (50 ml of 5 M)
top up to 500 ml and autoclave
5M Potassium Acetate
49.0g in 100ml of ddH20 and autoclave
20% SDS
20g in 100ml ddH20 and sterile filter
Resuspension Buffer
5l 1 M Tris
2l 0.5 M EDTA pH 8.0
to 10 ml ddH2O and autoclave
3M Sodium Acetate (trihydrate)
40.8lg in 100ml ddH20
pH to 7.0 with acetic acid and autoclave
Solutions for Alfalfa Fluorescent in situ Hybridization
3:1 Fixative:
3:1 - 100% ethanol : glacial acetic acid
Enzyme Mix:
Prepare working tubes of 1ml aliquots (stored at -20ºC):
750µl Cellulase (3% Cellulase)
250µl Pectolyase (1% Pectolyase)
note: pectolase is a combination of pectinases.
Formamide Denaturation Solution:
70% formamide
30% 2x SSC
Sodium Hydroxide Denaturation Solution:
0.05M NaOH/2x SSC
add - 47.5ml 2x SSC
- 2.5ml of 1.0M NaOH
Solutions for Tobacco Protoplast Expression
K3S Media
Chemical / For 1L / For 2L / [Stock] / Stock prep / [Working] / Working 1Sugar
sucrose / 136.9g / 272.8g / 0.4M
Salts
KNO3 / 10ml / 20ml / 100 / 62.5g/250ml / 24.73µM / 2.5g/L
MgSO4•7H2O / 10ml / 20ml / 100 / 6.25g/250ml / 1.01µM / 0.25g/L
NaH2PO4•H2O / 10ml / 20ml / 100 / 3.75g/250ml / 1.09µM / 0.15g/L
(NH4)2SO4 / 10ml / 20ml / 100 / 3.35g/250ml / 1.01µM / 0.134g/L
CaCl2•2H2O / 10ml / 20ml / 100 / 22.5g/250ml / 6.12µM / 0.9g/L
m-Inositol / 10ml / 20ml / 100 / 2.5g/250ml / 0.56µM / 0.1g/L
K3 Supplement
(microelements) / 10ml / 20ml
NH4NO3 / 10ml / 20ml / 100 / 6.0g/250ml / 3.0µM / 0.24g/L
FeSO4•EDTA / 5ml / 10ml / 200
Buffer
MES / 10ml / 20ml / 0.5M
pH5.7-5.8 / 5.0µM
Hormones
NAA / 1ml / 2ml / 1mg/ml
BA / 200µl / 400µl / 1mg/ml
Vitamins
B1 / 1ml / 2ml / 10mg/ml
B6 / 100µl / 200µl / 10mg/ml
NSA / 100µl / 200µl / 10mg/ml
Notes:NAA (-naphthaleneacetic acid)
B1 (thiamine hydrochloride)
B6 (pyridoxol hydrochloride)
NSA (nicotinic acid C6H6N2O MW122.13)
Adjust final pH to 5.7 – 5.8 via diluted KOH
Filter sterilize and store in 40 ml aliquots (store excess in -20ºC)
K3M Media
Chemical / For 1L / For 2L / [Stock] / Stock prep / [Working] / Working 1Sugar
sucrose / 30g / 60g
mannitol / 73g / 146g
Salts
KNO3 / 10ml / 20ml / 100 / 62.5g/250ml / 24.73µM / 2.5g/L
MgSO4•7H2O / 10ml / 20ml / 100 / 6.25g/250ml / 1.01µM / 0.25g/L
NaH2PO4•H2O / 10ml / 20ml / 100 / 3.75g/250ml / 1.09µM / 0.15g/L
(NH4)2SO4 / 10ml / 20ml / 100 / 3.35g/250ml / 1.01µM / 0.134g/L
CaCl2•2H2O / 10ml / 20ml / 100 / 22.5g/250ml / 6.12µM / 0.9g/L
m-Inositol / 10ml / 20ml / 100 / 2.5g/250ml / 0.56µM / 0.1g/L
K3 Supplement
(microelements) / 10ml / 20ml
NH4NO3 / 10ml / 20ml / 100 / 6.0g/250ml / 3.0µM / 0.24g/L
FeSO4•EDTA / 5ml / 10ml / 200
Buffer
MES / 10ml / 20ml / 0.5M
pH5.7-5.8 / 5.0µM
Hormones
NAA / 1ml / 2ml / 1mg/ml
BA / 200µl / 400µl / 1mg/ml
Vitamins
B1 / 1ml / 2ml / 10mg/ml
B6 / 100µl / 200µl / 10mg/ml
NSA / 100µl / 200µl / 10mg/ml
Notes:NAA (-naphthaleneacetic acid)
B1 (thiamine hydrochloride)
B6 (pyridoxol hydrochloride)
NSA (nicotinic acid C6H6N2O MW122.13)
Adjust final pH to 5.7 – 5.8 via diluted KOH
Filter sterilize and store in 2 ml aliquots (store excess in -20ºC)
Enzyme Solution
Chemical / Supplier / Concentration / For 100mlEnzyme / Calbiochem
Cellulase / Cellulysin Cat# 219466 / 0.5% / 0.5g
Macerase / Macerase Cat# 441201 / 0.2% / 0.2g
Media
K3S / 100ml
Prepare fresh and sterile filter (store excess in -20ºC)
FeSO4•7H2O Solution
Chemical / For 250ml / 200x Stock / 1x working / [working]FeSO4•7H2O / 1.39g/250ml / 27.8mg/L / 0.1µM
Na2EDTA•2H2O / 1.86g/250ml / 3.2mg/L / 0.1µM
Filter sterilize
K3 Supplement (microelements)
Chemical / For 500ml / 100x Stock / 1x Working / [Working]KI / 37.5mg/500ml / 0.75mg/L / 4.52µM
MnSO4•H2O / 500mg/500ml / 10mg/L / 59.16µM
ZnSO4•7H2O / 100mg/500ml / 2mg/L / 6.96µM
H3BO3 / 150mg/500ml / 3mg/L / 48.52µM
Na2MoO4•2H2O / 12.5mg/500ml / 0.25mg/L / 1.03µM
CoCl2•6H2O / 1.25mg/500ml / 0.025mg/L / 0.11µM
CuSO4 / 1.25mg/500ml / 0.025mg/L / 0.15µM
Filter sterilize (store excess at 4ºC)
25% PEG
Chemical / For 20ml / [Working]PEG 6000 / 5.0g / 25%
mannitol / 1.64g / 0.45M
Ca(NO3)2•4H2O / 0.47g / 0.1M
pH to 6.0
Filter sterilize and store in 2 ml aliquots (store excess in -20ºC)
W5 Washing Solution
Chemical / For 1LCaCl2•6H2O / 27.4g
or
CaCl2•2H2O / 18.4g
NaCl / 9.0g
KCl / 0.4g
Glucose / 1.0g
0.5M MES pH 5.7-5.8 / 10ml
Filter sterilize and store in 40 ml aliquots (store excess at -20ºC)
GUS/LUC Extraction Buffer
Chemical / For 100ml (from stock) / [Stock] / [Working]Na2HPO4 pH 7.0 / 5ml / 1M / 50mM
EDTA (Na2) pH 8.0 / 2ml / 0.5M / 10mM
N-laurylsacrosine (Na salt) / 286µl / 35% / 0.1%
Triton X-100 / 1ml / 10% / 0.1%
Add 1µl/ml of β-mercaptoethanol (10mM) just before use (store excess in -20ºC)
GUS Reaction Buffer
Chemical / concentrationGUS/LUC Extraction Buffer
MUG
4-Methylumbelliferyl β-D-glucuronide
sigma cat#: M-5664 / 22mg/25ml
Add 1µl/ml of β-mercaptoethanol (10mM) just before use
Note: MUG is light sensitive – wrap in foil.
Solutions for Western Blots
Western Blocking Solution 1
25 mM Tris-HCl (pH 7.5) - (for 1 L, 25 ml of 1 M)
150 mM NaCl - (for 1 L, 50 ml of 3 M)
5% (w/v) dried milk powder
0.2 % Tween-20
Western Blocking Solution 2
25 mM Tris-HCl (pH 7.5) - (for 1 L, 25 ml of 1 M)
150 mM NaCl - (for 1 L, 50 ml of 3 M)
0.2 % Tween-20
Western Blot Transfer Buffer
2.21 g CAPS
100 ml methanol
900 ml ddH2O
pH to 10.6 via KOH
store at 4°C
Western Mg2+Wash Buffer
100 mM Tris HCl - (for 1 L 12.11 g )
4 mM MgCl2 - (for 1 L 0.813 g)
pH to 9.8
Western Detection Solution
In 15 ml of western Mg2+wash buffer add,
16 l BCIP
20 l NBT (nitroblue tetrazolium)
Solutions for the Mangonel DNA Extraction
Homogenizing Buffer (50ml)
10.0ml 1M Tris-HCl pH 7.4
2.5ml 0.5M EDTA
2.5ml 5M NaCl
32.5ml dH2O
2.5ml 10% SDS (add last to avoid precipitation)
Sterile filter
*Do not use buffer more than 30 days old.
SHK Growth Medium(Components are given in mg/L)
Macronutrients
NH4H2PO4 300
KNO3 2500
CaCl2 2H2O 200
MgSO4 7H2O 400
K2SO4 4350
Micronutrients
KI 1
H3BO3 5
MnSO4 H2O 10
ZnSO4 7H2O 1
Na2MoO4 2H2O 0.1
CuSo4 5H2O 0.2
CoCl2 6H2O 0.1
Na2 EDTA 20
FeSO4 7H2O 15
Amino Acids
Proline 288
Thioproline 53
Vitamins
Myo-inositol 200
Nictonic acid 5
Pyridoxine HCl 0.5
Thiamine HCL 5
Other
Kinetin 0.2
Sucrose 30000
Agar 6000
pH 5.8
1