Hi,

Using zirconia 1-2 mm beads (1g) in 2 ml screw cap tubes, add tissue and buffer, with or without dipping in liquid N2, use 6000 rpm for 40 s in a Roche Magna lyser. It will give nice results. This is a very good method for homogenization. Good luck.

Thangasamy

Please check that your Nanodrop or UV-Vis spectrophotometer and your method of quantification are alright. Yield will always be low (and quality will be high!) for  a kit, but with Qiagen one can not doubt the kit. Also, for A. thaliana (standardized methods and trouble free small leaves!) there should not be a trouble unless there is a horrible mistake somewhere. For example, you say the lysis buffer is AP1, but I can remember it is either RLT or RLC depending on the case for Qiagen DNeasy Plant Mini Kits. Please check back and let's communicate. Thanks, Biswa

the kit wont give you good quality of DNA (A. thaliana) using kit. best is to do it maually not with kits. you simply crush the leaf samples in extraction buffer  and procedd with purification and precipitations. theres a lot of literature on manual DNA extraction from arabidopsis. i have undergone the sample trouble with qiagen DNA extraction kit and ended up with getting huge amount of good DNA by manual buffer making and manual extraction, u can try. try manually..

I am very much agree with Dr. Praveen Guleria. You can use CTAB buffer to extract the genomic DNA. Take about 1 g of A. thaliana fresh leave, wash it thoroughly and grind in Pestle and mortar with 1 ml of CTAB - extraction buffer. Also use RNase A and Proteinase K in the following steps to remove the RNA & protein and by precipitation step get rid-off the polysaccharides. All the steps should be done gently otherwise DNA will get sheared.

Hi Brijesh i am sending you the whole procedure for Plant DNA Isolation

PLANT DNA ISOLATION:

Principle – CTAB is a cationic detergent, which solublizes membranes and forms a complex with DNA. Proteins are extracted using chloroform/isoamyl alcohol and the CTAB-DNA complex is precipitated using/ with isopropanol. The DNA pellet is washed in alcohol and then dried and redissolved in T₁₀E₁ buffer. RNase and ammonium acetate precipitation is followed to remove RNA and polysaccharides.

Materials used:

1.                   Liquid nitrogen (it has temperature of -196°C).

2.                   Isolation buffer or DNA isolation buffer.

It is made up of following chemicals.

s.no.

Chemicals

stock concentration

Working concentration

for 100ml working solution

1

Tris HCl (pH-8)

1M

100Mm

10ml

2

EDTA (pH-8)

0.5M

20mM

4ml

3

NaCl

4M

1.4M

35ml

4

CTAB

10%

2%

20ml

5

Water

-

-

-

6

Β-mercaptoethanol

2%

0.2%

200µl (add 2µl/ml to the DEB just before use)

3.                   Chloroform-isoamyl alcohol is 24:1.

4.                   RNase solution is 10mg/ml RNase in 10mM. (Tris HCl, 15mM NaCl (pH-7.5), boiled for 15 minutes, cooled to room temperature and stored at -20°C).

5.                   100% isopropanol.

6.                   Washing solution 70% ethanol, 10mM ammonium acetate.

7.                   TE buffer, 10mM tris HCl and 1Mm EDTA (pH-8).

       FUNCTIONS OF THESE CHEMICALS :

1.       chloroform:isoamyl alcohol (24:1) is used to remove proteins from preparations of nucleic acids.

2.       Chloroform denatures the proteins and facilitates the separation of the aquous and organic phases.

3.       Isoamyl alcohol reduces foaming produce during extraction. The phenol:chloroform :isoamyl alcohol mixture be stored under 100mN.

4.       SDS (sodium dodecyl sulphate) acts as detergent agent which dissolves the cell wall by distructing the lipids surface tension.

5.       0.5M EDTA (pH-8)- ethylene diamine tetraacetate chelates divalent cations (Ca and Mg pectate of cell wall).

6.       β mercaptoethanol is an antioxidant which prevents oxidation of DNA by polyphenols.

7.       NaCl of 1.4M concentration removes histone proteins from the DNA thus we get pure DNA without histone protein.

8.       Liquid nitrogen- facilitates grinding and decreases the temperature of materials so cellular enzymes are not able to act over nucleic acids (DNA or RNA).

9.       RNase remove RNA from the DNA.

10.   Ammonium acetate removes polysaccharide.

11.   Isopropanol precipitates the DNA-CTAB complex.

PROTOCOL/ PROCEDURE :

1.       Grind up to 3g of fresh or 0.5g of lyophilized or dried plant material to a fine powder, using liquid nitrogen (absolute alcohol) mortar and pestle. Liquid nitrogen is not essential for lyophilized tissue, but facilitates the grinding procedure. Fresh tissue should not be thaw/grinded before being added to the isolation buffer, since cellular enzymes may rapidly degrade the DNA.

2.       Transfer the powder into 15ml of (pre-warmed at 60°C) DNA isolation buffer in capped polypropylene tube. Suspend the clumps using spatula.

3.       Incubate for 30-60 minutes at 60°C in water bath. Mix by gentle swirling every 10 minutes.

4.       Add 1 volume that means equal volume of chloroform:isoamyl alcohol, cap the tube and extract for 10 minutes on a rotary shaker or by hand. Mixing should be done gently but thoroughly enough to ensure emulsification of the phases.

5.       Centrifuge for 10 minutes (5000g, room temperature) or for 15 minutes at room temperature. Centrifugation separates two different layers, upper layer is aqueous layer.

6.       Re-extract the aqueous phase (in this aqueous part DNA is present) once with fresh chloroform:isoamyl alcohol and centrifuge again.

7.       Transfer the aqueous phase to another polypropylene centrifuge tube using a large bore pipette.

8.       Add (0.7) volume (2/3 part) of ice cold isopropanol and mix gently but thoroughly by inverting the tube several times. If the DNA-CTAB complex precipitate by centrifugation (5000g, 10 minutes preferably at 4°C / 15000 rpm, 10 minutes).

9.       Add 20ml of washing solution 4°C, 70% alcohol solution agitate the pellet for a few minutes and collect by centrifugation (15000rpm at 4°C).

10.   Invert the tubes and drain on a paper towel for about 1 hour. Then put it for overnight drying after covering with parafilm with tiny holes. These should neither contain residual alcohol nor should these be too dry. In both the cases re-dissolving might be difficult.

11.   Add an appropriate volume of TE buffer and allow the pellets to dissolve overnight (at 4°C) without agitation.

12.   Add 0.5 volume of 7.5M ammonium acetate solution mix and chill on ice for 15 minutes.

13.   Centrifuge for 30 minutes (10,000g at 4°C). Transfer supernatant to a new tube, ad 2 volumes of 96% ethanol, mix inversion and store for 1 hour at 20°C.

14.   Centrifuge for 10 minutes (5000g, 4°C) wash pellet in 70% ethanol and centrifuge again. Discard the supernatant dry the pellet and dissolve in appropriate amount of T₁₀E₁ buffer.

COMMENT:

1.       RNase treatment may be given after 7th step to a final concentration of 50/100 µg/ml.

2.       Whenever possible freshly harvested young leaves or hyphal material should be used. If secondary compounds present in leaves are problem, flower petals may be a useful alternative.

3.       For short period of time, fresh tissue may be stored on ice. Storage for longer time requires either freezing gel or anhydrous Calcium sulphate (dehydration in alcohol works well).

4.       This procedure can be used with very low amount of tissue (upto 300mg).

5.       The detrimental influence of polyphenols and their oxidation products may be concentrated by different strategies :-

i.      Inclusion of polyphenol absorbant such as Bovine serum albumin (BSA) and soluble polyvinyl pyrrolidone (eg- PVP-40) in the isolation buffer.

ii.     Inclusion of phenoloxidase inhibitors such as diethyldithiocarbamic acid (DIECA).

iii.    Inhibition of polyphenol oxidation by inclusion of antioxidants such as sodium concentration of β mercaptoethanol in the isolation buffer.

6.       Polysaccharides make DNA preparations highly viscous and inhibit the activity of restriction enzymes. Strategies to remove polysaccharide include :-

i.      Purification of crude DNA preparation by passage through ion exchange columns.

ii.     Precipitation of DNA with polyethylene glycol (e.g. PEG 8000) leaving polysaccharides in supernatant.

iii.    Exploiting the differential ethanol precipitation of DNA and polysaccharides from aqueous solution containing different salt concentrations.

iv.   Treatment of DNA precipitation with pectic enzymes.

7.       More general strategies to purify crude DNA preparations from undesirable contaminant include ultracentrifugation in Cesium chloride density gradient and preparative electrophoresis in low melting point agarose. Now a day availability of raisin/matrices based DNA can be purified by binding it to glass beads or silica particles.

8.       Controlled heating at 65-70°C not only lyses cell but also inactivates heat labile proteases and other proteins.

OBSERVATIONS AND PRECAUTIONS:

1.       Grinding stage

2.       Incubation at 60°C

3.       Chloroform-isoamyl miximg

4.       Centrifugation

5.       Collection of aqueous phase.

6.       Isopropanol precipitation of DNA.

7.       DNA quantification.

PREPARATION OF SOLUTIONS :

1.       1M tris pH-8------100mM

N₁V₁            =        N₂V₂

1X V₁            =       100 X 10¯³ 100

      V₁          =         10⁴ X 10¯³

      V₁          =         10ml

2.       0.5M EDTA pH-8------20mM

N₁V₁             =       N₂V₂

0.5XV₁         =       20 X 10¯³ X 100

        V₁        =         20 X 10¯³ X 100

                                    0.5

        V₁        =         200 X 10¯³ X 20

                      =       4000 X 10¯³

        V₁        =         4ml

3.       4M NaCl -------1.4M

N₁V₁       =             N₂V₂

4X V₁      =             1.4 X 100

     V₁       =             1.4 X 100              =             140         = 35ml

                                       4                                          4

                4.     10% CTAB ------ 2%

     4.       10% CTAB ------2%

                If 10% concentration then normality is = 1/10 (10/100 = 1/10)

                If 2% concentrated then normality is = 1/50 (2/100 = 1/50)

                N₁V₁       =             N₂V₂

                1/10 X V₁              =             1/50 X 100

                             V₁               =             2 X 10    = 20ml

Hello,

The best way is not using kit.Crush the leaves after washing with buffer using mortar and pestle.This will helps you get genomic DNA from the leaves without fail.

1. I use a smaller mortar and pestle for grinding. Before grinding, the mortar and pestle are frozen by adding liquid nitrogen into the mortar. Although the instruction asks for 100 mg leaf tissue, I use 120 mg, because I know I will lost some of the powder after grinding. Use an autoclaved specula (cold) to carefully collect the powder into a tube. Then, follow the protocol exactly, you should get gDNA.

2. It depends on your applications; for PCR; you should have enough gDNA as template.

Hi, I have always used the Qiagen DNeasy Plant Mini kit and have no problem with it. I think the main issue here is to get the DNA out from the leaf tissue, and the first few steps in the protocol determine if you will get DNA in the end. A few modifications would be helpful:

1. Apart from cooling mortar and pestle with liquid nitrogen, you should also pre-cool the spatula and eppendorf tube before transferring the fine leaf powder (Note: it has to be fine powder, no bits and pieces of leaf tissue should be seen. To do this, add liquid nitrogen 2-3 times in between grinding. This will prevent sample from thawing. Do not allow the sample to thaw because the DNA will be degraded. OD reading can't tell whether the DNA is intact or not, you have to run it on agarose gel to check the integrity). 

2. Instead of using 100mg of wet weight as recommended by the protocol, try increasing it to 200mg or even 300mg because you will lose some during grinding and transferring. 

3. It is good to pre-heat buffer AP1 at 65 degree Celsius before adding it into the fine powder, as this will facilitate the release of leaf DNA into the buffer. Also, make sure you vortex the sample 2-3- times during incubation at 65 degree Celsius. You should not see any clumps at this stage, everything has to be homogenise in AP1 buffer.

These are the most critical steps when extracting DNA using Qiagen kit. After this, follow the protocol and you should get enough DNA! I always get around 50 - 80ng/ul of DNA. Good luck!

I do agree with Deepak Rajpurohit ·

You can use liquid nitroger or even without liquid nitrogen you can do the same in a motor and pastel (This is for plant tissue)

Hi,

Using zirconia 1-2 mm beads (1g) in 2 ml screw cap tubes, add tissue and buffer, with or without dipping in liquid N2, use 6000 rpm for 40 s in a Roche Magna lyser. It will give nice results. This is a very good method for homogenization. Good luck.

Thangasamy

mix the sample with LIQUID NITROGEN and Polyvinylpolypyrrolidone (PVPP) in a mortar, then transfer the mixture to a buffer tube with AP1 plus RNAse,

and follow the protocol

Thank you everyone for the answers. I tried TissueLyser from Qiagen and got the results. So, probably I was making a mistake in crushing. I think, I was crushing the leaves for a long time in order to make it fine and that was causing problems. Also, I was using between 80-100 mg leaves. So may be I was loosing some of the material in the mortar and pestle also. But using the TissueLyser gave very good results. Thanks again for the answers.

hydrogen sulphide is use for this