Difference between revisions of "PhiX:SsDNA isolation"

Latest revision as of 16:39, 22 February 2010

Full credit to Bentley Fane for base protocol.

Day One: phage amplification

(prepare 1M MgCl₂)

• prepare two times 60ml LB + 600ul 1M MgCl₂ + 300ul CaCl₂ in 250ml conical flasks (~10/5 mM MgCl₂/CaCl₂)
• add 1.2ml overnight C122 culture to each
• follow until .3 < OD_600nm < .4 (~1.5 hours)
• add 10ul glycerol stock (wt phage) to each
• plateau OD of both (we have two to check this)
• to +4^oC O/N

(phage will attach to cell debris o/n; fix shaker platform; check stuff for tomorrow)

Day Two: phage detachment

(prepare BE buffer)

• spin chilled lysate for 10 minutes, 2700g, 4^oC
• discard supernatent
• resuspend pellet in 2ml BE
• shake at 4^oC O/N

(phage will detach from cell debris in presence of EDTA in BE)

Day Three: ssDNA preparation

(check BE pH - Roche pronase says pH7.5 is nice + Tris)

• spin chilled lysate for 10 minutes, 2700g, 4^oC
• transfer supernatent to new tube, discard pellet
• add 20ul 0.5M EDTA (pH 8; to 10mM), 50ul 20% SDS (to 0.5%)
  • note: could add 20ul 1M CaCl₂ (to 10mM) - DIDN"T
• add 50ul 20mg/ml pronase (to 0.5mg/ml)
• incubate at 40^oC for 2.5 hours
• transfer to phase-lock tube (5PRIME)
• add 2ml phenol (Tris equilibrated, pH 7.8, with antioxidant) and mix
• spin for 2 minutes at 2500g
• decant upper layer into new phase-lock tube
• add 1ml phenol and 1ml chloroform and mix
• spin for 2 minutes at 2500g
• decant upper layer into new phase lock tube
• add 2ml chloroform and mix
• spin for 2 minutes at 2500g

Nanodrop spec and gel run for purity and concentration