View source for PhiX:SsDNA isolation

==Day One: phage amplification==
(prepare 1M MgCl<sub>2</sub>)
* prepare two times 60ml LB + 600ul 1M MgCl<sub>2</sub> + 300ul CaCl<sub>2</sub> in 250ml conical flasks (~10/5 mM MgCl<sub>2</sub>/CaCl<sub>2</sub>)
* add 1.2ml overnight C122 culture to each
* follow until .3 < OD<sub>600nm</sub> < .4 (~1.5 hours)
* add 10ul glycerol stock (wt phage) to each
* plateau OD of both (we have two to check this)
* to +4<sup>o</sup>C O/N
(phage will attach to cell debris o/n; fix shaker platform; check stuff for tomorrow)

==Day Two: phage detachment==
(prepare [[PhiX:Recipes | BE buffer]])
* spin chilled lysate for 10 minutes, 2700g, 4<sup>o</sup>C
* discard supernatent
* resuspend pellet in 2ml BE
* shake at 4<sup>o</sup>C 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<sup>o</sup>C
* 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 CaCl<sub>2</sub> (to 10mM) - DIDN"T
* add 50ul 20mg/ml pronase (to 0.5mg/ml)
* incubate at 40<sup>o</sup>C for 2.5 hours
* transfer to phase-lock tube (5PRIME)
* add 2ml phenol (Tris equilibrated, pH 7.8, with antioxidant: see [[PhiX:Recipes]]) 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