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Q: Bacillus subtilis genome DNA extraction protocol?
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W. C. Lee
Jan 4, 2003, 2:04:15 AM1/4/03
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Dear b.m.methds-reagnts readers,
Do anyone know the easy and quick protocol to
extract genomic DNA from Bacillus subtilis?
I used a plant genomic DNA extraction kit from
Qiagen, which was the only kit available to me,
and couldn't get my PCR done.
Thanks in advance,
Woo
Mikhail Sinev
Jan 6, 2003, 10:25:13 AM1/6/03
to
I used QIAamp DNA mini kit(51304) for this goals.Ensure lysis is
complete.20h of lysis works, but I didn't play with time. 2h incubation was
bad. I had problem with PCR anyway,but resolve it using Vent polymerase
instead of fancy ones ("proofreading"). Taq always works good, but ...My
genes were too long to use Taq. Check G/C% of your oligos. Sometimes it is
difficult to find enough G/C in Bacillus.
ES
complete.20h of lysis works, but I didn't play with time. 2h incubation was
bad. I had problem with PCR anyway,but resolve it using Vent polymerase
instead of fancy ones ("proofreading"). Taq always works good, but ...My
genes were too long to use Taq. Check G/C% of your oligos. Sometimes it is
difficult to find enough G/C in Bacillus.
ES
"W. C. Lee" <aa1...@mail.ecc.u-tokyo.ac.jp> wrote in message
news:3E16876F...@mail.ecc.u-tokyo.ac.jp...
kavita...@lycos.com
Feb 4, 2003, 4:39:58 AM2/4/03
to
hi ,
i use a modified protocol for routine genome extraction from Bacillus..and works with a very good reproducibility.
1. firstly wash n pellet cells (10 ml culture)
2. resuspend cells in 1 ml TES buffer(sucrose 25%=EDTA 0.005 M+tris Cl pH 7.5 0.05M)..incubate at 65C for 1 hr.
3. add lysozyme..incubate at 37C for 15 min
4. add proteinase K (50 ug/ml) ...incubate at 37C for 15min.
5. follow routine phenol chloroform extraction n then precipitation.
this is if u can do without a kit else...Gentra systems offers a kit called "Puregene" which should do wonders since DNA quality is great!!!
Regards,
Kavita.
i use a modified protocol for routine genome extraction from Bacillus..and works with a very good reproducibility.
1. firstly wash n pellet cells (10 ml culture)
2. resuspend cells in 1 ml TES buffer(sucrose 25%=EDTA 0.005 M+tris Cl pH 7.5 0.05M)..incubate at 65C for 1 hr.
3. add lysozyme..incubate at 37C for 15 min
4. add proteinase K (50 ug/ml) ...incubate at 37C for 15min.
5. follow routine phenol chloroform extraction n then precipitation.
this is if u can do without a kit else...Gentra systems offers a kit called "Puregene" which should do wonders since DNA quality is great!!!
Regards,
Kavita.
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