Difference between revisions of "PhiX:SsDNA isolation"

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''Full credit to Bentley Fane for base protocol.''
 +
 
==Day One: phage amplification==
 
==Day One: phage amplification==
 
(prepare 1M MgCl<sub>2</sub>)
 
(prepare 1M MgCl<sub>2</sub>)
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==Day Two: phage detachment==
 
==Day Two: phage detachment==
(prepare BE: see [[PhiX:Recipes]])
+
(prepare [[PhiX:Recipes | BE buffer]])
 
* spin chilled lysate for 10 minutes, 2700g, 4<sup>o</sup>C
 
* spin chilled lysate for 10 minutes, 2700g, 4<sup>o</sup>C
 
* discard supernatent
 
* discard supernatent
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* incubate at 40<sup>o</sup>C for 2.5 hours
 
* incubate at 40<sup>o</sup>C for 2.5 hours
 
* transfer to phase-lock tube (5PRIME)
 
* transfer to phase-lock tube (5PRIME)
* add 2ml phenol (Tris equilibrated, pH 7.8, with antioxidant: see [[PhiX:Recipes]]) and mix
+
* add 2ml phenol ([[PhiX:Recipes |Tris equilibrated, pH 7.8, with antioxidant]]) and mix
 
* spin for 2 minutes at 2500g
 
* spin for 2 minutes at 2500g
 
* decant upper layer into new phase-lock tube
 
* decant upper layer into new phase-lock tube

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 < 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