Difference between revisions of "PhiX:Recipes"
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| − | '''BE Buffer ( | + | '''HIGH-SPEED GEL ELECTROPHORESIS'''<br> |
| + | '''SB Buffer'''<br> | ||
| + | 45g boric acid<br> | ||
| + | 8g sodium hydroxide pellets<br> | ||
| + | water to 1 litre | ||
| + | |||
| + | |||
| + | '''PHAGE ELUTION'''<br> | ||
| + | '''BE Buffer (50mM Na<sub>2</sub>B<sub>4</sub>O<sub>7</sub>; 5.0mM EDTA)'''<br> | ||
19.1g Na<sub>2</sub>B<sub>4</sub>O<sub>7</sub>.10H<sub>2</sub>O<br> | 19.1g Na<sub>2</sub>B<sub>4</sub>O<sub>7</sub>.10H<sub>2</sub>O<br> | ||
1.86g EDTA (disodium salt)<br> | 1.86g EDTA (disodium salt)<br> | ||
water to 1 litre | water to 1 litre | ||
| + | |||
| + | '''PHAGE GROWTH'''<br> | ||
| + | '''LB Buffer'''<br> | ||
| + | 10g tryptone<br> | ||
| + | 5g yeast extract<br> | ||
| + | 10g NaCl<br> | ||
| + | water to 1 litre, mix and autoclave<br> | ||
| + | after autoclaving add CaCl<sub>2</sub> to 2mM (2ml 1M per litre) and MgCl<sub>2</sub> to 10mM (10ml 1M per litre) | ||
| + | |||
| + | ''For plate agar:'' | ||
| + | add 15g Bacto brand agar per litre; | ||
| + | no need to add salts | ||
| + | |||
| + | ''For top agar:'' | ||
| + | add 7g Bacto brand agar per litre; | ||
| + | after autoclaving add CaCl<sub>2</sub> (to 2mM) and MgCl<sub>2</sub> (to 10mM) | ||
| + | |||
| + | ''Note:'' | ||
| + | Bentley Fane add CaCl<sub>2</sub> to 5mM | ||
| + | |||
| + | |||
| + | '''PHAGE STORAGE/DILUTION'''<br> | ||
| + | '''HFB Buffer (60mM NH<sub>4</sub>Cl; 90mM NaCl; 0.1M KCl; 0.1M Tris-HCl (pH 7.4); 1.0mM MgSO<sub>4</sub>; 1.0mM CaCl<sub>2</sub>)'''<br> | ||
| + | ''For 500ml 10× stock:''<br> | ||
| + | 125ml of 2.0 M Tris-HCl (pH 7.3) -- surely 250ml?<br> | ||
| + | 15g NH<sub>4</sub>Cl<br> | ||
| + | 2.5g NaCl<br> | ||
| + | 25g KCl<br> | ||
| + | 5.0ml 1.0 M MgSO<sub>4</sub><br> | ||
| + | 5.0ml 1.0M CaCl<sub>2</sub><br> | ||
| + | water up to 500ml<br> | ||
| + | |||
| + | ''For working solution:'' | ||
| + | dilute to 1× in water and autoclave | ||
| + | |||
| + | |||
| + | '''NUCLEIC ACID SEPARATION'''<br> | ||
'''Tris-saturated Phenol with antioxidant (with 100ml phenol)'''<br> | '''Tris-saturated Phenol with antioxidant (with 100ml phenol)'''<br> | ||
add 100mg 8-hydroxyquinoline (i.e., 0.1% w/v)<br> | add 100mg 8-hydroxyquinoline (i.e., 0.1% w/v)<br> | ||
| Line 9: | Line 54: | ||
add 130ul 2-mercaptoethanol (0.2% of buffer phase) | add 130ul 2-mercaptoethanol (0.2% of buffer phase) | ||
| + | |||
| + | '''COMPONENT SOLUTIONS'''<br> | ||
'''EDTA stock (0.5M)'''<br> | '''EDTA stock (0.5M)'''<br> | ||
use disodium salt and pH to 8 with NaOH | use disodium salt and pH to 8 with NaOH | ||
| Line 21: | Line 68: | ||
dissolve in nuclease-free water and store at -20<sup>o</sup>C | dissolve in nuclease-free water and store at -20<sup>o</sup>C | ||
| − | ''' | + | '''10 X PE1'''<br> |
200mM Tris pH 7.5<br> | 200mM Tris pH 7.5<br> | ||
100mM MgCl<sub>2</sub><br> | 100mM MgCl<sub>2</sub><br> | ||
| Line 27: | Line 74: | ||
10 mM DTT | 10 mM DTT | ||
| − | ''' | + | '''10 X PE2'''<br> |
| − | 200 mM Tris | + | 200 mM Tris pH 7.5<br> |
100 mM MgCl<sub>2</sub><br> | 100 mM MgCl<sub>2</sub><br> | ||
100 mM DTT | 100 mM DTT | ||
Latest revision as of 05:14, 7 August 2013
HIGH-SPEED GEL ELECTROPHORESIS
SB Buffer
45g boric acid
8g sodium hydroxide pellets
water to 1 litre
PHAGE ELUTION
BE Buffer (50mM Na2B4O7; 5.0mM EDTA)
19.1g Na2B4O7.10H2O
1.86g EDTA (disodium salt)
water to 1 litre
PHAGE GROWTH
LB Buffer
10g tryptone
5g yeast extract
10g NaCl
water to 1 litre, mix and autoclave
after autoclaving add CaCl2 to 2mM (2ml 1M per litre) and MgCl2 to 10mM (10ml 1M per litre)
For plate agar: add 15g Bacto brand agar per litre; no need to add salts
For top agar: add 7g Bacto brand agar per litre; after autoclaving add CaCl2 (to 2mM) and MgCl2 (to 10mM)
Note: Bentley Fane add CaCl2 to 5mM
PHAGE STORAGE/DILUTION
HFB Buffer (60mM NH4Cl; 90mM NaCl; 0.1M KCl; 0.1M Tris-HCl (pH 7.4); 1.0mM MgSO4; 1.0mM CaCl2)
For 500ml 10× stock:
125ml of 2.0 M Tris-HCl (pH 7.3) -- surely 250ml?
15g NH4Cl
2.5g NaCl
25g KCl
5.0ml 1.0 M MgSO4
5.0ml 1.0M CaCl2
water up to 500ml
For working solution: dilute to 1× in water and autoclave
NUCLEIC ACID SEPARATION
Tris-saturated Phenol with antioxidant (with 100ml phenol)
add 100mg 8-hydroxyquinoline (i.e., 0.1% w/v)
add equilibration buffer (-> pH 7.9) - 6.5ml of this in Sigma kit
add 130ul 2-mercaptoethanol (0.2% of buffer phase)
COMPONENT SOLUTIONS
EDTA stock (0.5M)
use disodium salt and pH to 8 with NaOH
SDS 20%
pre-bought - avoids fine powder
Tris-HCl stock (1M)
pH to 8 with HCl
Pronase to 20mg/ml
dissolve in nuclease-free water and store at -20oC
10 X PE1
200mM Tris pH 7.5
100mM MgCl2
200 mM NaCl
10 mM DTT
10 X PE2
200 mM Tris pH 7.5
100 mM MgCl2
100 mM DTT
