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-   -   DNA extraction from Tissue (http://www.molecularstation.com/forum/dna-forum/83438-dna-extraction-tissue.html)

moleculargeek21 08-30-2011 07:17 PM

DNA extraction from Tissue
 
Im starting to get a bit frustrated with this wasted effort with little to show for it. Im extracting DNA from human tissues, liver, pancreas, etc. The equipment Im using for the extraction is the FUJI Film autogen nucleic acid isolator. My procedure is as follows:
Start with 5-10 mg of tissue (in this case liver)
Add 180 microliters of MDT tissue lysis buffer
Add 20 microliters of EDT Proteinase K

This is allowed to incubate at 55 C on a rotary shaker until dissolved.
Sample is centrifuged at 8000 rpm for 3 minutes at room temperature
180 microliters of LDT lysis buffer is added
Vortexed for 15 seconds
incubated in water bat at 70 C for 10 minutes
240 microliters of > 99% 200 proof ethanol is added
Vortexed for 15 seconds
Sample is loaded and extracted.
The sample was analyzed using nanodrop 2000
The nanodrop was blanked with Tris EDTA 10mM1mM
Analysis:
Nucleic acid concentration: -30.6 ng/ul
A260:-.613
A280:-.521
260/280: 1.18
260/230: .44
Any help would be greatly appreciated.

danfive 08-31-2011 05:17 PM

Re: DNA extraction from Tissue
 
Quote:

Originally Posted by moleculargeek21 (Post 435905)
I’m extracting DNA from human tissues, liver, pancreas, etc.
1.Start with 5-10 mg of tissue (in this case liver)
2.Add 180 microliters of MDT tissue lysis buffer
3.Add 20 microliters of EDT Proteinase K
4.This is allowed to incubate at 55 C on a rotary shaker until dissolved.
5. Sample is centrifuged at 8000 rpm for 3 minutes at room temperature
6.180 microliters of LDT lysis buffer is added
7.Vortexed for 15 seconds
8.incubated in water bat at 70 C for 10 minutes
9.240 microliters of > 99% 200 proof ethanol is added
10.Vortexed for 15 seconds
11.Sample is loaded and extracted.
12.The sample was analyzed using nanodrop 2000
The nanodrop was blanked with Tris EDTA 10mM1mM
Analysis:
Nucleic acid concentration: -30.6 ng/ul
A260:-.613
A280:-.521
260/280: 1.18
260/230: .44
Any help would be greatly appreciated.

OK first I don't know this Fuji extraction systems or it's buffers--but I'll give my tips anyways.

1st. Nanodrop zeroing may be off (TE may or may not be appropriate). Easy fix is to dilute DNA solution 1:10 or 1:20 into TE--and voila--now blank should work very accurately.

2nd. I numbered your protocol and see a few problems (pretty big list actually) are these manufacturer's instructions or someone's specific protocol?

*5. Sample is centrifuged at 8000 rpm for 3 minutes at room temperature/6.180 microliters of LDT lysis buffer is added--OK what is going on here?
If you do ProK digestion--it is common to centrifuge--remove supernatant to new 1.5ml tube--quench pro-K (boiling or buffer inactivation)--pellet in original tube, if any, is waste.

* 7.Vortexed for 15 seconds
8.incubated in water bat at 70 C for 10 minutes
---This is all cool and shit--probably de-activating the Pro-K, but its important that the centrifuged pellet (may not be visible) from step 5 is removed from the supernatant before vortexing in step 7.

*9.240 microliters of > 99% 200 proof ethanol is added
10.Vortexed for 15 seconds
11.Sample is loaded and extracted.
--These steps are very necessary for pelleting your DNA, but some things are omitted. The sequence should be
a. add Ethanol (can range from 70-99%),
b. mix (vortex or invert tubes),
c. Centrifuge to Precipitate DNA pellet at max (~14,000xg) for 10-20 minutes,
d. Remove solution (mostly Ethanol) from DNA pellet (may be very small speck at bottom of tube, or sizable pellet),
e. dry for ~10min to rid of remaining EtOH, then dissolve pellet in 100ul H2O, TE buffer,
f. allow to go into solution for ~10min at RT or ON at 4C., go to nanodrop procedure

I don't understand #11, if that is a spin column procedure--that's cool, it will capture the DNA, seperate it from the EtOH, and elute in a suitable buffer. Just again make sure that a bunch of cell debris is not in there (referring to #5) cuz it will clog the spin column.

moleculargeek21 08-31-2011 06:25 PM

Re: DNA extraction from Tissue
 
I carried out another extraction this afternoon, my spectral data appears more promising.

Nucleic Acid Concentration: 111.8 ng/ul
A260: 2.235
A280: 1.138
260/280: 1.96
260/230 1.67

baseline correction of 340 nm
blanked with nuclease free water
I did not carry out any Rnase treatment.

Saoirse 01-17-2012 06:15 PM

Re: DNA extraction from Tissue
 
I have used the fujifilm many times and your protocol is fine...except it doesn't say if you mix your sample after adding the LDT....otherwise the protocol is exactly as it is written in the book....so I would guess it was your nanodrop that was the issue...blank using the fujifilm elution buffer also run your sample on a gel to see what it looks like

csxw 06-02-2012 01:31 AM

Re: DNA extraction from Tissue
 
so I would guess it was your nanodrop that was the issue...blank using the fujifilm elution buffer also run your sample on a gel to see what it looks lik

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