DNA Extraction

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magicsiew
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DNA Extraction

Post by magicsiew » Thu May 20, 2010 7:24 am

Hi, I am using QIAamp kit to extract DNA from Acanthamoeba culture. However, I just able to extract about 40ng/ul of concentration only when measured by NanoQuant. Is this concentration normal for Acanthamoeba ? This is because previously extraction from other sample can obtain high concentration up to 300ng/ul. Or is there any pre-treatment required to be done to the sample before using this Qiagen kit? I am sure there is no other step stated in Qiagen handbook, just that any other step that is specifically to acanthamoeba?

goonwolf420
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Post by goonwolf420 » Thu May 20, 2010 8:01 am

How many cells are you using for DNA extraction? From my experience you need around 150,000 cells to get decent concentrations (i.e. > 100ng/ul)

magicsiew
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Post by magicsiew » Thu May 20, 2010 8:14 am

I pippete 3ml from the culture. Is that enough?

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JackBean
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Post by JackBean » Thu May 20, 2010 12:12 pm

what is your OD600 or other cell count?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Thu May 20, 2010 1:10 pm

I did not do cell count, just pipette it from the culture. What is the density of normal OD600 or cell count required to get normal DNA concentration yield?

magicsiew
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Post by magicsiew » Fri May 21, 2010 6:04 am

I have done cell count, using 3.2million culture, but i just pipetted 1ml and do the extraction with the modified protocol. Add in proteinase K initial, incubate at 56'c for 10min. Yield positive results.

magicsiew
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Post by magicsiew » Fri May 21, 2010 6:05 am

After that follow the protocol provided as in QIAamp handbook*

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