I'm trying to extract genomic DNA from E. coli using phenol:chloroform extraction method (as suggested by http://www.bio-protocol.org/e97). How do I ensure that the overnight bacteria culture contains the sufficient titre of bacteria (planned to use 1.5 mL out of 10 mL cultured broth) in order to get a yield of DNA up to 5 to 10 micrograms? Do I need even need to have mid-log phase bacteria to proceed with the extraction (by doing 100 fold dilution for once and check for OD)?
Thank you.
If your final OD is around 2.5 to 3 then 1.5ml should be enough. Mid log phase culture may not give you that yield. And if its an overnight culture, you will have to dilute atleast 10 times to take an OD.
That 1.5 mL out of 10 mL itself is actually the overnight culture bacteria. Do I still need to dilute it for 10 times as well?
If I really have to, is it going to be 100 fold for each?
1.5ml of the 10ml overnight culture can be used for isolating the DNA without diluting provided the OD is around 2 - 3.
Dilution is recommended only for taking OD. 10 times would suffice. take 100microliter from the 10ml stock and make it up to 1ml using the same media used for blank and take OD.
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