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dna isolation from bacterial cultures


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#1 dhanashree prabhu

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Posted 10 March 2015 - 11:35 PM

hello this is dhanashre,

i have been trying to extract DNA from broth grown cultures of marine bacteria but everytime i do it i get good results upto the nanodrop measurement step, when i run the DNA in a gel i get streaks over the band or get dragged bands, i tried doing the phenol-chloroform -isoamyl alcohol method which involved bead beating but then we thought that the bead beating might be brekaking the DNA so we quit that and used lysozyme for cell lysis but that resulted in very very low yield of DNA, i also tried cutting the tips of my pippette tips to avoid DNA breakage but got similar results, i will b carrying out DNA extraction using proteinase K and phenol-chloroform-isoamyl alcohol, i keep my DNA for ethanol extraction overnight and then for solubilization in TE buffer overnight, any care tips that i should take in order to get higher concentration of intact genomic DNA?



#2 phage434

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Posted 11 March 2015 - 06:16 AM

That sounds like overloaded gels, rather than poor DNA. How much DNA are you loading on your gel?

In general, you don't need high molecular weight DNA for most purposes. PCR and next gen sequencing can both use DNA which is fragmented to the 50 Kb size with no difficulty.



#3 dhanashree prabhu

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Posted 11 March 2015 - 11:04 PM

i have been told to add 2microlitres of DNA sample plus 8 or 10 microlitres of 6X gel loading buffer....



#4 phage434

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Posted 12 March 2015 - 05:23 AM

Well, if you are already only doing what you are told, why are you asking for advice? The idea is to learn how to think, not to be a cheap robotic pipettor.



#5 Leishman001

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Posted 12 March 2015 - 07:19 AM

Hi dhanashree:

 

Try this protocol from the March 2015 issue of Biotechniques. It seems tailored to what you are trying to isolate (genomic DNA) from bacteria.

Link: http://www.biotechni...ues-357170.html

 

The article is new so I haven't tried the protocol, but Biotechniques has a good reputation. I haven't had a bad experience with the protocols that they choose to highlight.

 

Alternatively, try a commercial kit to extract your genomic DNA. Qiagen makes a good kit (QIAamp DNA mini kit) that works well for extracting full length viral DNA, so it should also work for genomic. Qiagen also posts the recipes for most of their buffers so you can make your own buffers and continue your preps more cheaply.

 

Keep an eye on your A260/A280 ratios when you are checking your DNA on Nanodrop. The correct ratio for DNA should be below 1.9. A higher ratio points to RNA contamination. That might contribute to your streaky bands.

 

You probably already know this, but don't vortex your samples after cell lysis. Vortexing will shear your genomic DNA.

 

Good luck and don't get discouraged. It takes a few tries to get it right sometimes.

 




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