isolation of genomic bacterial DNA (bacillus) (View forum version)



Hira Muzzamal_27321

Posted 28 November 2011 - 10:32 AM

Hi to all,
i am working on bacillus strains and extracting their DNA by standard Phenol/chloroform extraction method using SDS and CTAB/NaCl, but i'm unable to isolate good, unsmeared and darker DNA bands. i have also even changed the concentrations and quantities but still no success.Using CTAB method was also not successful.
please anyone can suggest a protocol for isolating good quality DNA from Bacteria specially Bacillus.
thanks...
H Muzzamal

phage434

Posted 28 November 2011 - 05:21 PM

Key for isolation in gram + bacterial is cell lysis, which may not be adequate with SDS and CTAB. An enzyme step may be required with lysozyme, mutanolysin, or similar enzymes. A smear is expected for well isolated genomic DNA and does not indicate a problem.

Hira Muzzamal_27321

Posted 29 November 2011 - 08:56 AM

thanks for ur answer. cell lysis was the main problem i was facing during the procedure.thats why i used the protocol
with and without SDS for DNA.but using CTAB with 2%marceptoethanol was a little helpful but again the only problem was
light DNA.now i'll try lysozyme in the protocol...
well thanks for help...:)