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Arabidopsis DNA Extraction from Leaves

Arabidopsis DNA Extraction from Leaves - Arabidopsis and Plant Biology

Arabidopsis DNA Extraction from Leaves - Discuss Arabidopsis and Plant Biology Research.


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  #1  
Old 11-11-2006, 10:05 PM
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Post Arabidopsis DNA Extraction from Leaves



Hey guys,

can I get some input on how to extract DNA from Arabidopsis leaves?

thank you
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  #2  
Old 04-25-2008, 09:09 AM
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Default Re: Arabidopsis DNA Extraction from Leaves

I have a good protocol that i use which is quite quick... unfortunatly its at uni so I'll have to post it on Monday for you.
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Old 04-25-2008, 05:51 PM
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Thumbs up Re: Arabidopsis DNA Extraction from Leaves

Here's a procedure I used in the past:

1. Weigh 0.1-0.2 grams tissue (~4 full trifoliate leaves) in 1.5ml microfuge tube. Quick freeze and grind to powder with blue pestle –don’t let thaw
2.Add 0.75ml EB buffer
EB buffer: 100mM Tris pH8.0
50mM EDTA pH8.0
500mM NaCl
10mM beta-Merc (add beta-merc fresh everytime)
3. Continue mixing until mixed well and thawed.
4. Add 0.05ml 20% SDS, shake well and incubate tubes at 65C 10min
5. Add 0.25ml 5M potassium acetate, shake well and incubate tubes on ice (0C) 20min. (Keep on ice until all samples are lysed).
6.Spin tubes at 21,000g for 20min
7.remove supernatant w/pipetman (~1ml) and put through mira cloth into new 1.5ml tube.
8. add 500ul isopropanol, invert to mix well and incubate at -20C for 30min-1hr.
9. pellet DNA at 21,000g for 15min—pour off supernatant and dry pellets by inverting tube on paper towel for 5min.
10. Redissolve pellets with 0.7ml TE (50mM Tris, 10mM EDTA pH8) by agitating; making sure pellet is resuspended.
11. Spin tube at max speed for 10min to remove insoluble debris.
12. Transfer supernatant to new tube and add 75ul 3M sodium acetate and 500ul isopropanol.
13. Mix well by inverting and spin to pellet DNA for 30sec.
14. Wash pellet with 700ul 80% EtOH—spin and remove EtOH, dry ~5min inverted on paper towel.
15. redissolve in 100ul 10mM Tris, 0.5mM EDTA pH 8.0.
16. Let sit ON at 4C.
17. Check OD260 and dilute for PCR (~1/100 or 5ng/ul).

Solutions.
750ul/sample—EB Buffer: 100mM Tris pH 8.0, 50mM EDTA pH 8.0, 500mM NaCl, 10mM beta-mercaptoethanol
50ul/sample—20%SDS
250ul/sample---5M Potassium acetate
700ul/sample---50mM Tris, 10mM EDTA pH 8.0
1mL/sample—Isopropanol
75ul/sample—3M NaAcetate
700ul/sample—80% EtOH
100ul/sample--10mM Tris, 0.5mM EDTA pH 8.0

Materials and incubators.
Blue plastic pestles.
-20C freezer
65C Water bath
4C refrigerator
Ice 0C
spectrophotometer
Mira Cloth
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Old 04-26-2008, 12:05 AM
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Default Re: Arabidopsis DNA Extraction from Leaves

The edwards prep seems to me to be much simpler... It does use 2 1.7ml tubes but doesnt require any fresh solutions.

Again will post it on monday for you.
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Old 04-30-2008, 01:50 AM
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Default Re: Arabidopsis DNA Extraction from Leaves

Hi!

For those of you who asked I have posted the protocol here:
[Only registered users see links. ]

The pdf version of it is available here:
[Only registered users see links. ]

Hope it helps someone!
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Old 04-09-2010, 05:42 AM
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Default Re: Arabidopsis DNA Extraction from Leaves

cant open this protocol....dont knw y....they sya the wb page has been expired....
can some one send this protocol to my e mail....
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