Difference between revisions of "PhiX:SsDNA isolation"
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==Day One: phage amplification== | ==Day One: phage amplification== | ||
(prepare 1M MgCl<sub>2</sub>) | (prepare 1M MgCl<sub>2</sub>) | ||
Revision as of 17:17, 18 August 2009
With special 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 < OD600nm < .4 (~1.5 hours)
- add 10ul glycerol stock (wt phage) to each
- plateau OD of both (we have two to check this)
- to +4oC 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, 4oC
- discard supernatent
- resuspend pellet in 2ml BE
- shake at 4oC 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, 4oC
- 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 40oC 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
