9 replies to this topic

[123blockhead]

Hi,

I have extract bacterial genomic DNA using phenol chloroform extraction method and did gel electrophoresis after DNA extraction.
However, there was no intact band observed, only smear as shown in the attached image.
Could anybody help me to analyze the possible reasons? Thanks.

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[Adrian K]

There will not be intact band shown in gel... the smears were RNA which you did not eliminate.

[123blockhead]

adrian kohsf, on 29 May 2011 - 08:39 PM, said:

There will not be intact band shown in gel... the smears were RNA which you did not eliminate.

is it there will be 1 intact band for genomic DNA that we extracted when we run gel?
because i searched online, got pic show very nice intact band.

if the smears were due to RNA, then where is my genomic DNA?
when i measure OD, i get pretty good A260/A280 ratio, 1.7-1.9.
this is the indication of DNA, no contamination of RNA, right?
(sorry, i'm stil new to lab, correct me if i'm wrong)

i'm gonna send the DNA for whole genome sequencing, so the DNA integrity is very important.  
now try to figure out what's wrong with my extraction.

Edited by 123blockhead, 29 May 2011 - 09:36 PM.

[123blockhead]

these are the few samples of genomic DNA that i extracted using Qiagen DNA mini kit.
there are some fluorescent in the well of the gel. is it the DNA stuck in the well and cannot be seperate out?
i'm using 1% gel in 1xTBE buffer, run in 90v for 1 hour.

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[Trof]

123blockhead, on 29 May 2011 - 09:24 PM, said:

i'm gonna send the DNA for whole genome sequencing, so the DNA integrity is very important.  
now try to figure out what's wrong with my extraction.

What kind of genome sequencing are you sending it for?
Do you know that most of current genome-sequencing techniques shredd the DNA into as small as 25 bp (or as large as 100 bp, unless there was some minor improvement over last few years) fragments that are sequenced separately and contigs then assembeled by a powered up computers?

[Adrian K]

IMHO, For your second gel (assume you totally eliminated the RNA)... the smear means some extend degradation of your genomic DNA...

A260/A280 ratio (from what I understand) only shows the purity of your DNA, but not degradation/fragmented of DNA, that might explain the smearing, but with good ratio value.

If your genomic DNA is very concentrated,  your well will shows bright (as in your second gel). This is because the genomic DNA will hardly go into the gel.

I'm not sure if your genomic dna is good for whole genomic sequencing... i never done that.

[123blockhead]

Trof, on 30 May 2011 - 06:00 AM, said:

123blockhead, on 29 May 2011 - 09:24 PM, said:

i'm gonna send the DNA for whole genome sequencing, so the DNA integrity is very important.  
now try to figure out what's wrong with my extraction.

What kind of genome sequencing are you sending it for?
Do you know that most of current genome-sequencing techniques shredd the DNA into as small as 25 bp (or as large as 100 bp, unless there was some minor improvement over last few years) fragments that are sequenced separately and contigs then assembeled by a powered up computers?

i'm sending for whole genome re-sequencing using illumina next generation sequencing.

i think they probably will fragmentize the DNA into 150-300bp?
they want to have intact genomic DNA so they can fragmentize the DNA by random. when they have 30x-50x coverage, probably is enough to cover all region of DNA and we just assemble the reads.

if the DNA already shear in the beginning, probably the DNA would have break in certain sites (or tends to break in certain sites), so maybe some region would not be sequenced.

[123blockhead]

adrian kohsf, on 30 May 2011 - 11:38 AM, said:

IMHO, For your second gel (assume you totally eliminated the RNA)... the smear means some extend degradation of your genomic DNA...

A260/A280 ratio (from what I understand) only shows the purity of your DNA, but not degradation/fragmented of DNA, that might explain the smearing, but with good ratio value.

If your genomic DNA is very concentrated,  your well will shows bright (as in your second gel). This is because the genomic DNA will hardly go into the gel.

I'm not sure if your genomic dna is good for whole genomic sequencing... i never done that.

they want to see the intact band like the attached pic below. i wonder why i couldn't get this kind of result. troubleshooting, troubleshooting, troubleshooting....  

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[Adrian K]

I not sure how big is your genomic DNA. If your size is more than 50kb, there is not much chance for your DNA to be able to enter to the gel in normal conditions.
I know you are not convinced. Try to look at the commercial ladders: http://www.fermentas...electrophoresis
The maximum they can produce is 48502bp. And run in agarose 0.4%.

If the sequencing company claimed the intact band can be achieve, I really interested to learn their method.

Edited by adrian kohsf, 31 May 2011 - 08:43 AM.

[123blockhead]

adrian kohsf, on 31 May 2011 - 08:38 AM, said:

I not sure how big is your genomic DNA. If your size is more than 50kb, there is not much chance for your DNA to be able to enter to the gel in normal conditions.
I know you are not convinced. Try to look at the commercial ladders: http://www.fermentas...electrophoresis
The maximum they can produce is 48502bp. And run in agarose 0.4%.

If the sequencing company claimed the intact band can be achieve, I really interested to learn their method.

The genomic DNA is around 5Mb. i have run on 0.5% gel.
Finally i got the intact band using phenol chloroform extraction even though there are little smear.
Don't know the sequencing company would accept or not.
But in terms of purity, the A260/A280 ratio is not very good yet.
hmmm...

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