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Old 06-15-2018, 01:14 AM   #1
_chiara_
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Default quality check 1 ng DNA

Hi everybody
I am willing to sequence with MiSeq using a Nextera XT library starting with very challenging samples of not cultivable bacteria.

I am now checking the quality of my DNA after extraction (I can get a maximum of 16 ul of extract only).

I managed to quantify it with a high sensitivity kit (QBit measurement) and I currently know I have 1 ng DNA.

Does anybody know any efficient way to visualise 1 ng of DNA on gel?
I tried the classical protocol with 1% agarose and ethidium bromide and of course I couldn't see anything...

Any insight on how to check purity and fragmentation on such small DNA quantities?

Thanks a lot
Chiara
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Old 06-15-2018, 02:28 AM   #2
nucacidhunter
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You can use Bioanlyser high sensitivity DNA Chip to visualize up to 12Kb and over (minimum 5pg) or gDNA ScreenTape up to 50kb (minimum 1ng). Either of these will indicate the size range suitable for XT library prep. If you do not have access to any, try library prep without quality check just by using correct quantity.
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Old 06-15-2018, 07:20 AM   #3
JBKri
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Hi Chiara,
it seems SybrGold is sensitive down to 25 pg of DNA, see https://www.thermofisher.com/gr/en/h...na-stains.html
According to this page, Picogreen is equally sensitive.
I imagine the dye used for the Qubit assays is equally sensitive.
We have Picogreen in the lab, we could try it.
Jon
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Old 06-15-2018, 07:42 AM   #4
ATϟGC
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If agarose gel electrophoresis is your only option for fragment size distribution, GelStar is also very sensitive and might be more suitable than Sybr Gold for using in the gel itself.

Gelstar use with a DarkReader:
http://www.clarechemical.com/gelstar.htm

I tried GelStar about 6 years ago with a free trial I got and it worked very well in my hands.
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Old 06-17-2018, 04:38 AM   #5
_chiara_
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Glad to see I have so many different options to check!

Thanks everybody
I'll keep you updated for the results!

Chiara
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Old 07-04-2018, 01:00 AM   #6
JBKri
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Quote:
Originally Posted by ATϟGC View Post
If agarose gel electrophoresis is your only option for fragment size distribution, GelStar is also very sensitive and might be more suitable than Sybr Gold for using in the gel itself.

Gelstar use with a DarkReader:
http://www.clarechemical.com/gelstar.htm

I tried GelStar about 6 years ago with a free trial I got and it worked very well in my hands.
Would anyone happen to have a pdf of this:
GelStar nucleic acid gel stain: high sensitivity detection in gels.
White HW1, Vartak NB, Burland TG, Curtis FP, Kusukawa N. Biotechniques. 1999 May;26(5):984-8.

Could PAGE gels potentially be more sensitive? Do they work with the same stains as agarose gels?

On a side note, I had a look at the gel images comparing GelStar with EthBr at Lonza, and if you look carefully you will see that they have in fact used the same image for both dyes. The Ethidium Bromide image is identical, just dimmed a little bit. You would think they could take the trouble to get some real images...

Jon
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fragmentation, gel electrophoresis, low starting material, purity, quality check

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