MYCOPLASMALOGY - MYCOPLASMA DNA EXTRACTION (Nov/09/2009 )

I'm planning to isolate DNA from MYCOPLASMA SP., but I have issues with this....

1. Since, I'm gonna use QIAGEN column method, there are possible chances that I might lose DNA, either due to irreversible binding of DNA to the column ALSO they might go undetected by the UV-SPEC.

2. I'm planning to use a carrier DNA to elute the Mycoplasma DNA, but the problem I'd face is the interference of CARRIER DNA during pcr amplification of these DNA's....

I NEED SOLUTIONS FOR THESE TWO QUESTIONS.

1. How to maximize the extraction of DNA using column method, with out using a carrier DNA?

2. If carrier DNA is used how to do the PCR with out the carrier DNA interfering the PCR?

I have more questions with reference to this experiment, but these are the basics.....

Please share your suggestions.....

Thanks in advance...

Hi there!

Maybe you can try another extraction kit. I know this one: PrepSEQ™ Mycoplasma Nucleic Acid Extraction kit

bioalex20 on Nov 17 2009, 09:54 AM said:

Are you working on Mycoplasma and related fields....if so please let me know....

Microbes in action on Nov 9 2009, 11:28 AM said:

I don't work on this issue, but I guess as long as you know the carrier dna sequence you may know the level of noise it'll give you in the PCR. I think that there are several software packages that let you guess were will this unspecific amplification may take place. Also you may use negative controls in which you run the carrier DNA without your target dna or with a different non-primer interacting dna plus the carrier. After that you can substract the noise (This would be easier in qPCR).

As for the elution I suppose you have tried things as high NACl,Urea,formamide. You may add electrophoretic power to help your DNA progress to the liquid phase.

Hey,

Thanks so much for your valid suggestions,

I've just done one part as to how the carrier DNA is influencing the standard curve for the Q-PCR......., and there is a complete shift of the standard line that is lograthamic,

I'm planning next to do a control in the absence of carrier DNA.

Well, would you mind to explain as to how to apply electrophoretic power in isolation of DNA.

if you have some article with reference to this, please do mail me.......

drselwynwraj@gmail.com

Microbes in action on Nov 17 2009, 12:47 PM said:

Sorry it was just a suggestion, but I've read that RNA electroelution is much more efficient than standard diffusion, which also makes sense for DNA. It's just like an in-column southern blot. I don't know about the column you use but the elution scheme should be rather simple:
Fullfill the column of liquid phase, take a copper cable from the - pole to where you inyect the sample and an other copper cable from the + pole to where you collect the sample. The voltage should be proportional to the column size respect to the voltage u use for southern blotting, due to the column resistance I would recommend using half of that obtained voltage to prevent column damage due to heat.

I've just found something that may give you a clue: http://www.ncbi.nlm.nih.gov/pubmed/9237572

Hey,

Thanks so much.....

Sounds interesting.....I'll give a try....

I'm not sure of your time zone...anyways I'll mail you in detail about my work......

I'll be happy if you can share your thoughts about it and the faesibility of work.....

Thanks.

Microbes in action on Nov 17 2009, 02:26 PM said:

Yep, certainly, my email: uo155943@uniovi.es