protocol for plant DNA isolation

[nanditajena]

i want to know the recent and accurate protocol for plant DNA isolation.

[danfive]

CTAB or Qiagen DNeasy Plant DNA extraction kit or similar from different company.

[yourlabdata]

I use a slightly modified edwards prep.

Much much much much easier than CTAB and much much much cheaper than quiagen kits... depending on how many preps you are doing extraction time can go as low as 2 minutes per prep and the dna is good enough to do preety much anything with... I havent done a quiagen kit in 3 years!

[yourlabdata]

Hi!

I use this protocol here:
[Only registered users see links. ]

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

Hope it helps!

[danfive]

Wow that Edwards protocol is extremely short. Some questions, how long do you expect your isolated DNA to last? Does it work on root tissue as well as leaves?


I've posted this protocol before, I used itfor gene mapping and produces stable DNA that would last 1-2 years at -20C.

Originally Posted by danfive

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

[yourlabdata]

Hi!!!

I still use dna that i extracted this way about 2 years ago and its fine... just make sure you use TE not water.

I have maped to genes using this protocol to extract DNA and have been able to, with practice, reduce the time per sample to about 2-3 minutes!!!

I strongly recomend it!!!

I have no idea if it works on roots.... Worth giving it a shot since its so quick!

Cheers,

Ed.

[abhijittoke]

hi,,,, thanks for ur data... will u plz mail me that one again? i need to use thet ... my id is [Only registered users see links. ]

[pselva7]

i cant get the edwards protocal through the link wat people were menioned earlier.. if anybody hav that protocal plz send to me my mail id [Only registered users see links. ]... thanks in advance...

[danfive]

Samples for PCR analysis (usually leaf tissue) are collected
using the lid of a sterile Eppendorf tube to pinch out a disc of
material into the tube. This ensures uniform sample size and also
reduces the possibilities of contamination arising from handling
the tissue. DNA is extracted as follows: The tissue is macerated
(using disposable grinders from Bel-art Products: Scienceware,
Pequannock, NJ, 07440 USA. catalog no 992) in the original
Eppendorf tube at room temperature, without buffer, for 15
seconds. 400 ,u of extraction buffer (200 mM Tris HCl pH 7.5,
250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the
sample vortexed for 5 seconds. This mixture can then be left
at room temperature until all the samples have been extracted
(> 1 hour). The extracts are centrifuged at 13,000 rpm for 1
minute and 300 ul of the supernatant transferred to a fresh
Eppendorf tube. This supernatant is mixed with 300 ul
isopropanol and left at room temperature for 2 minutes. Following
centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum
dried and dissolved in 100 ul 1x TE. This DNA is stable at 4°C
for greater than one year. 2.5 ul of this sample is sufficient for
a standard 50 ul PCR (Figure 1). When older tissue is used this
may be increased to 25 ul without any deleterious effect on the
PCR. Using this protocol we have found it possible to process
hundreds of individual samples in a single working day.
from Edwards K, Johnstone C, Thompson C: A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.
Nucleic Acids Res 1991 , 19:1349.

Here's a link to the article, 1 page ---> [Only registered users see links. ]

And here is a link to a modified Edwards protocol
---> [Only registered users see links. ]

and a quote

Combinations of DNA isolation protocols from Edwards et al. [9] and Walbot and Warren [10] were thus compared using ~2.5 mg (~3 mm2) of A. thaliana leaf tissue in 50 μl of extraction mixture, from which 1 μl was used as template in a routine PCR application. We have found that one of the variant extraction buffers, hereafter called Sucrose Solution, exhibited no change in the efficiency of PCR amplification judged by ethidium bromide-stained agarose gels in response to varying the pH from 7.0 to 8.0, or changing the salt and sucrose concentrations between 200 to 400 mM NaCl and 300 to 440 mM sucrose, respectively (data not shown). Due to the presence of sucrose in the extraction buffer, we named this nucleic acid extraction method the Sucrose Prep. In agreement with Thomson and Henry [11], we found that heating the crude extract for 10 min followed by centrifugation for 5 sec at 6000 g eliminated nearly all debris that interferes with PCR amplification.

[danfive]

Rapid DNA preparation


ftp://ftp.arabidopsis.org/home/tair/...manual/ch2.pdf

A crude and rapid extraction from a small leaf disk is sufficient to isolate genomic DNA suitable for PCR amplification. The present protocol is based on the method described by Edwards et al. (1991). All steps are performed at room temperature.

1. Use the lid of a 1.5 ml Eppendorf tube to pinch out a disc from a young leaf into the tube. Alternatively, add fresh roots cut from seedlings or plants (Note A).

2. Use a small pestle to grind the leaf material in the tube without buffer for approx. 15 seconds.

3. Add 400 μl of extraction buffer (Note B), and vortex for 5 seconds (Note C).

4. Spin 1 minute at full speed in a microfuge to pellet the debris.

5. Transfer 300 μl of the supernatant into a fresh 1.5 ml Eppendorf tube (avoid taking debris from the pellet..

6. Add 300 μl isopropanol, mix and leave at room temperature for approx. 2 minutes.

7. Spin 5 minutes at full speed in a microfuge to pellet the DNA.

7.extra step. Add 400 ul EtOH 80%, spin 5 min

8. Remove all the supernatant and dry the pellet gently (for instance by incubating the open tubes at 37°C for a few minutes, or speedvac for 2-3 min). Do not let the pellet get too dry, otherwise it will be very difficult to redissolve the genomic DNA.

9. Add 100 μl TE, and dissolve the pellet by gentle shaking (do not vortex).

Notes
A. It is important to avoid using too much starting plant material. In contrast, a smallerpiece of leaf or a single cotyledon can be used, in which case all volumes should be reduced by 50% in the subsequent steps of the extraction procedure.

B. Extraction buffer: 200 mM Tris-HCl pH 7.5 (pH 8.0 is OK), 250 mM NaCl, 25 mM EDTA, 0.5% SDS

C. At this stage, the samples can be kept at room temperature for up to one hour until all the samples have been extracted

D. TE: 10 mM Tris ph7.5, 1 mM EDTA



Edwards, K., Johnstone, C., and Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acids Res. 19, 1349.

From ---> [Only registered users see links. ]