发信人: shawl (this used to be my playground), 信区: Biology
标 题: Re: Help!!! BAC DNA isolation...
发信站: Unknown Space - 未名空间 (Sun Aug 22 15:45:46 2004) WWW-POST

1.start from 3-4ml fresh overnight culture, pellet cell in 8000rpm in cold for
3min
2.resuspend in 200ul P1
3.lysis with with 400ul P2 <5min in RT
4.neutralize with 300ul P3 on ice for 5 min
5.spin down 10min at maxi spped in cold
6.supernatant to a new tube
7.add 0.75ml cold isopropanol
8.spin at maxi speed RT for 15min
9.discard supernatant, wash with 0.5ml ethanol, spin 5min maxi speed in cold
10. asirate supernatant and air dry DNA till pellet starts to turn translucent
Caution: DON'T OVER DRY.


【 在 victorjenny (victor) 的大作中提到: 】
: I want to isolate BAC DNA and screen, but the problem now is that I couldn't
: isolate BAC DNA.
: I tried two ways, but failed.
: 1) Use QianGen miniprep kit, like what I did in plasmid miniprep.
: 2) Lysis with QianGen miniprep kit P1, P2,and N3, use 0.7 volume to
preciptate
: DNA, use 70% EtOH to clean.
: I got genome DNA all the time. :(
: There might be some BAC DNA, but restriction enzyme digestion only got
smear.
: Since I couldn't see any obvious band, I can't say there is BAC.
: Thank you for your advice in advance.
:


--
※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 128.249.]