- Transform E. coli cells with target plasmid DNA.
- Grow a 10 ml overnight culture of isolate in 2XYT + drugs
- Inoculate 50 ml of 2XYT with 1.0 ml of o/n culture
- shake @ 37C, 30 min. (250 ml flask)
- add 200 ul helper phage M13KO7 (6 x 10(11) pfu/ml)
- shake @ 37C, 30 min.
- add 70 ul Kanamycin (50 mg/ml) and continue shaking 10-14 h.
- Transfer cells to oak ridge tubes and pellet @ 17,000 g for 15 min. [ 10K rpm, swinging bucket rotor ]
- pour supernatant into a fresh tube and spin again 15 min.
- transfer the supernatant to a 50 ml graduated dispo-tube and add 1/4 volume 3.5M NH4oAc / 20% PEG solution
- invert to mix and pour into a fresh centrifuge tube
- leave on ice 30 min.
- centrifuge 15 min, 17,000g
- thoroughly drain the supernatant and resuspend the pellet in 200 ul TE3 (10mM Tris.Cl (8) / 1mM EDTA).
- transfer to an Eppendorf tube and add 100 ul buffer saturated phenol and 100 ul chloroform
- vortex for 1 full minute and spin 5 min. in a microfuge
- remove the top aqueous phase and transfer to a fresh tube. (leave behind the large interface)
- repeat the extraction until there is only a slight interface (5-6X)
- add 200 ul chloroform, vortex, and spin for 2 min.
- tranfer the aqueous phase to a fresh tube
- Precipitate ssDNA with 100 ul 7.5M NH4oAc and 600 ul Ethanol
- leave on mice for 30 min. Spin for 15 min at 4C
- drain the supernatant and carefully rinse the pellet with 95% EtOH
- dry in speed vac (no heat)
- dissolve pellet only in 20 ul H2O and transfer to a fresh tube
- Examine yeild on a gini mel - there should be no smaller forms
- Quantitate ssDNA
- dilute 5 ul of yeild to 1 ml in H2O (1:200)
- measure Absorbance of UV 260 & 280 nm
- assume 1 A260/ml = 40 ug/ml ssDNA