Hi Sreeram,

Please have a look for this video and protocol may help you!

Good luck

Ali

http://www.jove.com/science-education/10218/community-dna-extraction-from-bacterial-colonies

Thanks. I had seen this. But this one is again extracting from soil. Its for isolating nonculturable bacteria. My case is different, where I want to isolate from agar plates in large numbers.

Why you cant use an autoclaved glass rod and roll it tightly on the agar surface to get all the colonies stick to the rod? 

Alex: Of course I could use them to get the colonies out. My problem is getting the DNA off them after scraping. My question is about that.

Hi Sreeram,

We use a quick protocol to detect plasmid inserts directly from E. coli.

Pick a very small portion of colony with a sterile pipette tip or a toothpick and put it into a 10 ul of sterile distilled water. Heat it at 94 degree C for 5 min followed by cooling at 4 degree. Spin for 5 min at 10000 rpm. take sup. as template for PCR.

This has worked well practically with all the plasmid inserts in our lab.

I hope it should also work for 16s analysis also.

The only disadvantage is that the sup.  has to be used immediately, without any storage.

Alternatively, you can also try direct addition of part of colony to PCR mix. but sometimes it gives poor quality amplicon.

Hope this will help.

Dear Sreeram

Use any method like the classical phenol-chloroform method. You don't need to grow the colonies again.

 The polymerase chain reaction can be used for comparative analysis of various phages or for preparing sequencing templates. Phage DNA usually does not have to be purified prior to its amplification by PCR but can be amplified in situ using lysed phages. The protocol involves the following steps:

1.       Pick a phage plaque and suspend it in 50microliters of water, leaving it at room temperature for 30-60min to allow the phages to diffuse into the water.

2.       For 50 micro liters PCR amplification, remove 25 micro liters of the phage suspension to a clean tube and denature by heating in boiling water bath for few minutes.

3.       Mix gently, add reagents and proceed as prescribed by the vendors of the PCR reagents.

did you find a suitable protocol?

what's wrong with people putting up answers that aren't even remotely close to the question asked?!

Leonid: Yes exactly totally unrelated answers ! I gave up on replying to these comments after a while.... But thanks, I found a suitable protocol. Well, basically I scraped them all off and resuspended in lysis buffer and continued further steps the same was as I would with the pellet of an O/N culture. Worked fine.

Hi Sreeram, how did you scrap them all off if you have huge amount of them and you are doing a screening?Would you please to share more details?many thanks!

I think the best is to pick individual colony, restreak on another new plate and expand for couple of days, or then resuspend in liquid media for overnight. You can then later use the entire bacteria for DNA isolation. All the best

Hi Zia, Thanks for the input. I am collecting cells. It will be a cell based screening. Don't think I can pick colonies one by one.

If it is bacterial cell based, then I guess this should be a right approach, otherwise keeping in mind the huge possible variations in bacterial cell even in same population, I don't think you can compare it in future. In case of human/animals cells, I guess that would be possible.

Hi Bo Zhou,

Actually I dont have a separate protocol for this. But basically what I did was scraped off the colonies from a plate using pipette tip. Then I resuspended in sterile PBS buffer. Spun at 10000 rpm for 5 min. And followed lysed with lysis buffer, lysozyme treated the lysate followed by proteinase K treatment etc.... as per the protocol provided by the kit as you can get from the link below. I used the sigma bacterial genome elute kit:

https://www.sigmaaldrich.com/technical-documents/protocols/biology/genelute-bacterial-genomic-dna-kit.html

It worked for me. Hope it clears your question. All the best! Bo Zhou

Cheers, Sreeram

Thank you Zia and Sreeram !

http://www.dartmouth.edu/~staphy/protocol/fast_colony_dna_extraction.html

Let me at the very onset tell you that 16s DNA sequencing is not accepted by many reviewers as the homology alignment often gives wrong results. You will find several of such reports on the internet. Nevertheless, you do not need such massive cell mass to do this work, just some picogram of cells are more than sufficient. If you are interested, you may just grow the organism on a suitable agar slope and pour some sterile saline, shake well and whatever cell comes off is more than sufficient for the work. Of course these cell must be centrifuged to remove all media components and saline components and then suspend it in the appropriate buffer and proceed. Good luck.

Hello , universal, cheap, simple, easy and reliable method for isolation DNA.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310651/