12. Transfer the
DNA solution to a 1.5ml microcentrifuge tube and add 700 µl of phenol:chloroform, vortex gently until the phases mix, spin for 5 minutes (microfuge top speed, room temperature) and pipette the top layer into a clean microcentrifuge tube. Be sure to not transfer the white interphase.
13. Extract once more with 700 µl of phenol:chloroform: vortex gently until the phases mix, spin for 5 minutes and pipette the top layer into a clean microcentrifuge tube. Be sure to not transfer the white interphase.
14. Add 700 µl of chloroform, vortex gently until the phases mix, spin for 5 minutes and pipette the top layer into a clean microfuge tube, being sure not to transfer any white interphase.
15. Add 700 µl of isopropanol, vortex gently until mixed and spin for 20 minutes. Discard the isopropanol, being sure not to lose the pellet. Wash the pellet with 500 µl of 70% ethanol, remove excess by pipeting and air-dry for 30 minutes.
16. Dissolve the pellets in 100 µl of TE, pH 8.0.
17. Quantitate on the flourometer (use pico green) or spectromphotometer. Should get 4-7µg per 250ml culture that is 95-98% BAC
DNA.
18. Check for E. coli, by loading 2 ml on a 0.8% agarose gel in TAE, run > 63 V for 1 hour.
Reagent List------------
2X YT (Quality Biologicals, 340-023-100)
Glycerol stock: 400µl 50% glycerol and 400µl culture
10mM EDTA, pH 8.0
10% SDS
5N NaOH
** 7.5M potassium acetate (Sigma, P-3542)
Glacial acetic acid
RNase A (heat inactivated) (LTI or Sigma)
Isopropanol
10mM Tris/50mM EDTA, pH 8.0
50mM Tris/50mM EDTA, pH 8.0
** Phenol:chloroform:IAA(25:24:1) (Sigma, P-2069)
Chloroform
** indicates reagent should be from a specific company (not tested for relevance by us)
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