5 replies to this topic

[jehane]

Hello all

we face a problem concerning qualitative analysis of fragmented DNA
we extract DNA from liver of mouse and load the DNA sample on agarose gel but we didn't detect clear band.
photo is attached.

PLEASE Helppppppppppppppppp

Attached thumbnail(s)

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[dnafactory]

What did you exactly do? Did you extract DNA and load it onto the gel directly?

[jehane]

dnafactory, on Jun 17 2009, 03:39 AM, said:

What did you exactly do? Did you extract DNA and load it onto the gel directly?

at first homogenize liver tissue on TES lysis buffer
then take 100 ul of the homogenate to new eppendorf adding 50 ul proteinase k - incubate at 37 C with shaking overnight
then load sample onto the gel

[dnafactory]

jehane, on Jun 17 2009, 12:45 PM, said:

at first homogenize liver tissue on TES lysis buffer
then take 100 ul of the homogenate to new eppendorf adding 50 ul proteinase k - incubate at 37 C with shaking overnight
then load sample onto the gel

Than, this is the correct image of your DNA. You see a smear when you treat this way. If you use DNase or you sonicate, you will see some bands...

[jehane]

dnafactory, on Jun 17 2009, 04:09 AM, said:

jehane, on Jun 17 2009, 12:45 PM, said:

at first homogenize liver tissue on TES lysis buffer
then take 100 ul of the homogenate to new eppendorf adding 50 ul proteinase k - incubate at 37 C with shaking overnight
then load sample onto the gel

Than, this is the correct image of your DNA. You see a smear when you treat this way. If you use DNase or you sonicate, you will see some bands...

Using DNase or RNase??
sonication will affect the fragmentation?????????????????/

[dnafactory]

jehane, on Jun 19 2009, 04:53 PM, said:

dnafactory, on Jun 17 2009, 04:09 AM, said:

jehane, on Jun 17 2009, 12:45 PM, said:

at first homogenize liver tissue on TES lysis buffer
then take 100 ul of the homogenate to new eppendorf adding 50 ul proteinase k - incubate at 37 C with shaking overnight
then load sample onto the gel

Than, this is the correct image of your DNA. You see a smear when you treat this way. If you use DNase or you sonicate, you will see some bands...

Using DNase or RNase??
sonication will affect the fragmentation?????????????????/

Well, when you sonicate you shear the DNA and you areable to see distinct bands. If you do not sonicate, you usually see a lot of DNA in the well and a smear. Using micrococcus DNase will have the same effect of sonication.
As for "normal" images of DNA you can have a look here: www.biomedcentral.com/1471-2202/6/13/figure/F2