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從λ噬菌體DNA的純
點(diǎn)擊次數(shù)：1052 發(fā)布時(shí)間：2016-2-29
This is a modification of the procedure in Short Protocols (1-41,1-45)
1. Prepare a 50 ml lIQuid lysate:
A. mix 2xlO8 E. coli Cells with 100 ul phage (from one picked plaque in 500 ul SM), and
100 ul lOmM MgCl2/lOmM CaCI2. Adsorb at 37° for 15 min.
B. transfer to 50 ml LB medium (supposed to be NZC, we"ve used NZCYM successfully), shake well at 37° for 6 to 8 hrs (can be done overnight with no obvious difference in DNA yield).
C. add 375 ul CHC13 and shake for 15 min.
D. Transfer to round-bottom polypropylene tube, leaving behind the chloroform.
E. Centrifuge 10 min @10,000 rpm in 870 rotor on IEC at 4°.
F. Decant supeRNAtant to 50ml conical bottom tubes. Add a few drops chloroform if the
lysate is to be stored at 4°. Otherwise, proceed to part II.
H. DNA isolation from lysate:
A. Add 10AI 5 mg/ml DNase I and 25 fil 10 mg/ml DNase-free RNase. incubate one hour
(according to Short Protocols) or overnight @ 37".
B. If the solution has become cloudy (may happen in overnight incubation), centrifuge 10 min in 15 ml conical tubes in clinical cfuge at top speed. Transfer supernatant to polyallomer tubes and centrifuge 1.5 hr at 27,000 rpm at 4" in SW 28.1 rotor (have to divide into 3 tubes). (See also alternate procedure.)
C. Resuspend phage pellet in 180 141 5OmM Tris, ph 8, 20 141 TES (100 mM Tris pH 8, 100 mM EDTA, 1 % SDS). Add 20 pLI of 10 mg/ml proteinase K and incubate one hour (can be left overnight) at 37".
D. Add 1 vol of buffered phenol. Mix well and microcentrifuge 2 min. Transfer aqueous layer to new tube, extract with phenol again. You can do a third phenol extraction if there is still a lot of white denatured protein at the interface.
E. Add 200 III CHC"3, shake, microcentrifuge briefly. Transfer aqueous layer to new tube,
repeat.
F. Add 20 jil of 3M Sodium Acetate pH 4.8 and 2 vol of room temperature 100% ethanol.
Mix, microcentrifuge 10 min to precipitate DNA.
G. Add 1 ml 70% ethanol and centrifuge 5 min. Dry in speed vac about 5 min, dissolve in
100 pl TE. Yield of DNA should be around 2OItg per 17ml ultracentrifuged media.
Alternative protocol for lambda DNA preparation:
After step II A (RNase and DNase digestion) add 2.9 g NaCl and 5 g PEG-8000 to each 50 ml tube (be careful to exclude clumps of PEG-8000, which are hard to dissolve.) Shake well to dissolve. Put on ice in 4" for 4 hours to o/n.
Centrifuge 20 min @3700 rpm in conical bottom 50 ml tubes in Chris Frazier"s tabletop cfuge.
Proceed as in step II C, except double volumes.
Then proceed to step D.
This procedure may inexplicably result in some RNA contamination. May add 20ttl of 10mg/ml DNase-free RNase and incubate one hour to overnight at 37" to remove it. Yield of DNA is around 20OIAg per 50 ml culture.