Difference between revisions of "PhiX:Recipes"

Latest revision as of 04:14, 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 -20^oC

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