The isolation of Bacterial DNA generally is well done on both colony or broth but form the colony is recommended. For colony, there is no need for kits, boiling is enough with high centrifugation and used the upper phase that contains the DNA. for, Broth the kits are recommended.  

I have kits for DNA isolation from bacteria , but i was wondering which is more recommended and would there be a negative effect on PCR of any of them? Moreover, do you have any experience in anaerobes and do they need special tricks in DNA isolation.

I had extracted DNA from bacterial cells (aerobic or non- ) and clinical samples using the Nucleospin® kit (MACHEREY-NAGEL, Germany), following the manufacturer’s instructions. Briefly, 1.5 ml of the bacterial culture or 1 ml of liquid sample was transferred into an Eppendorf tube and centrifuged at 5,000 rpm for 10 min. DNA was extracted from the pellets by resuspending the pelleted cells in 20 mM Tris/HCl; 2 mM EDTA; 1% Triton X-100; pH 8 supplemented with 20 mg/ml lysozyme and incubating for 30 min at 37 °C. Thereafter 25 μL of Proteinase K was added and samples were incubated at 56 °C until complete lysis was obtained. The DNA was eluted off of the NucleoSpin® Tissue Column by adding 100 μl prewarmed Buffer BE (70°C). Thus, I recommend that you try this procedure

I would recommend you to grow bacteria in a agar plate and then select an individual isolated colony and grow this colony in a broth. There are several fold advantages;

1. The bacterial cells grown in the broth will be from the pure culture with similar genetic characteristics (not a quasi-species picked up from the agar plate)

2. The yield of DNA will be much higher (high number of bacterial cells and they are not in clumps like grown in the agar)

3. Hence, higher yield of the DNA recovered can be used for multiple experiments and other molecular studies with the same batch of DNA extracted. Most of the time experiments fail and we need to repeat the experiment for which we need higher amount of DNA. We start the genetic analysis using the DNA extracted in first batch, finish few genetic analysis and the DNA. For further analysis we again extract DNA from the bacterial cells grown from culture stock and we are not sure whether the grown bacterial cells are the same strains. I just believe that there are quasi-species and this can affect your subsequent genetic analysis.

:-)

Hope this will be helpful

Regards

Thank you very much Mahmoud and Badri...

I did the DNA isolation for C.difficile and B. bifidium from broth :

Before DNA isolation, i centrifuged the tube containing bacterial culture , decanted the broth and freezed the cell pellet .(is this okey?)

Then using a QIAGEN -QIAamp DNA Mini kit i isolated DNA and got on average 150ng/ul concentration.

However , when i loaded the DNA , it was degraded (attached a gel photo) and i couldn't get a product for my PCR using both DNAs. 

Its good that you have loaded the gel picture. Is this the gel picture of the genomic DNA or the PCR product. Looking at the picture this looks like a gel picture of the sheared genomic DNA.

If this is a genomic DNA then you need to improve the DNA extraction. Please follow the steps of the DNA isolation kit manufacturer tightly. In addition following things would improve this;

1. Use of a freshly grown culture. I do not think there is an issue with the storage of the pellets.

2. Storage of the DNA in the medium or buffer. Just sterile distilled water is good enough.

3. Use of same TAE buffer for too long (multiple gels).

let me know if it improves.

Regards

God day,

I see you have troubles regarding:

1) PCR conditions may not be enough optimized, especially  annealing T. of primers

 2) Enzyme potency

3) Volumes of PCR materials should be rechecked

4) The used primers may not specific for targeting bacteria 

5) Preparation of gel (agarose concentration, voltage rate also the similarity of buffer used in preparation of gel and during the running process)

Please revised uppers points again with update if possible

i will repeat DNA isolation using 24hr cultures instead of 48hr cultures for both C.difficile and B.bifidium and see what will happen. 

Mahmoud the gel attached is for the gDNA and not for PCR (sorry if it wasn't clear mentioned). I am attaching another gel photo for the gradient PCR for both bacteria. The expected size product for B.bifidium is 424bp and for C.difficile is 157bp (i couldn't get any of them

Good morning  Suheir Nassar,

Sorry to see you faced difficulty in get positive results that will be corrected soon, I understand the gel photo for gDNA and yielded no bands. This finding revealed that there is no DNA extracted or present in very low level which unable detected by gel. Thus, try measure the DNA by nanodrop apparatus or spectrophotometer if possible.

If nanodrop gives positive , re-PCR testing with new primers sets and Promega PCR master mix (efficient) using colony samples instead of broth for DNA extraction and see what will happen

Regards,

Mahmoud

Generally speaking it tends to be easier to lyse broth-cultured bacteria than colonies. This is because fresh broth cultures are lessed stressed by the crowding, nutrient depletion and buildup of waste products that occurs in colonies. This might be particularly problematic with Bacillus or Clostridium strains which may form endospores which are extremely refractory. The ideal culture for DNA extraction is a mid-exponential phase broth culture. 

Thank you a lot Prof. Andrew for your explanation and information.

Prof Andrew..Is there  a different protocol to follow for  DNA isolation from anaerobic bacteria  than aerobes due to their special respiratory mechanism? or the difference in protocol is just for bacteria being Gram + ve or Gram - ve as in most kits' protocols?

You mentioned 'Before DNA isolation, i centrifuged the tube containing bacterial culture , decanted the broth and freezed the cell pellet'. Why did you freeze the pellet? This could have damaged the DNA, degrading it to smaller fragments, hence the gel image. Once you have a dry pellet you can resuspend in buffer, boil for 10mins to lyse cells then seperate cell debris from DNA by centrifuge.