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Pseudomonas aeruginosa DNA extraction


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#1 meedomada

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Posted 06 May 2015 - 02:08 PM

I read in some papers that genomic DNA can be extracted by simple boiling ( which looks very easy). I am not sure if this is  a good method compared to extraction kits. Could any one help re this issue, as I actually have an extraction kit from which I lost the RNase, and that is why I cannot use it.



#2 bob1

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Posted 06 May 2015 - 03:07 PM

The question here is: what do you want to do with the DNA? If for instance, you were just amplifying a gene by PCR, then boiling might be enough, but other applications might  require much higher quality DNA, in which case you could use a kit or almost any genomic DNA extraction procedure.



#3 meedomada

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Posted 07 May 2015 - 03:03 AM

Many thanks

Yes I will amplify some genes by PCR, so I guess will be ok then

But I read many techniques, in some they just do boiling, and in others they add some TE buffer!! so which is better?



#4 bob1

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Posted 07 May 2015 - 01:21 PM

Boiling basically just ruptures the cell walls so that the DNA and other cellular components are just floating around in a "soup". This soup can cause inhibition of the PCR, damage to the DNA and all sorts of problems for other methods such as cloning. Usually boiling would be done in a buffered solution, such as TE, which contains EDTA (metal ion chelator) and will hopefully inhibit many DNA damaging enzymes, as these usually require metal co-factors.

 

Other extraction methods lead to much cleaner DNA, which will last longer once extracted and are less likely to lead to problems for down-stream applications, but are still likely to end up suspended in TE, which preserves the DNA by buffering the environment so that acid hydrolysis of the DNA doesn't occur, and the EDTA prevents things growing in the solution.



#5 meedomada

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Posted 08 May 2015 - 10:16 AM

Many Thanks.

In that the extraction kit is much better even for PCR, but I am having the issue of the missing RNase, can I use it without the RNase?



#6 bob1

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Posted 09 May 2015 - 12:36 AM

Yes, but you can also buy RNases and they are incredibly stable (will renature after autoclaving...) so if you find an old vial in the lab, it will most likely still work.



#7 pstils

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Posted 09 May 2015 - 01:25 PM

I routinely run PCR on p.aeruginosa, and don't bother with extraction.  I coloney pick using a 10µL tip into 80µL water, use 1µL of that for template in the PCR and just add a 10 minute 95˚C step before whatever PCR I'm doing.  Works like a charm.



#8 meedomada

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Posted 09 May 2015 - 02:49 PM

Yes, but you can also buy RNases and they are incredibly stable (will renature after autoclaving...) so if you find an old vial in the lab, it will most likely still work.

Does this mean that by autoclaving RNase it becomes active again? I will look for some.

 

the problem is that ordering RNase will take months

 

I routinely run PCR on p.aeruginosa, and don't bother with extraction.  I coloney pick using a 10µL tip into 80µL water, use 1µL of that for template in the PCR and just add a 10 minute 95˚C step before whatever PCR I'm doing.  Works like a charm.

This sounds very easy and interesting, I will try it and hope it will work



#9 bob1

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Posted 10 May 2015 - 12:54 AM

Yes, but you can also buy RNases and they are incredibly stable (will renature after autoclaving...) so if you find an old vial in the lab, it will most likely still work.

Does this mean that by autoclaving RNase it becomes active again? I will look for some.
 
the problem is that ordering RNase will take months

No, autoclaving won't make it active again, just that RNases can re-fold after being through an autoclave, as an example of their robustness.

Ordering shouldn't take months... where are you getting it from/delivering it to?You can get RNase H from Sigma-Aldrich that will ship today/tomorrow.




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