Hi

have you add RNAses? if you had, may be it's due to DNA degradation during process

Long genomic DNA  are highly susceptible to shearing.  Did you used a silica column based method?

This is after RNAase treatment and clean up after the treatment. No i haven't used silica based method. i just used custom protocol

Check purity of the elutes. Protein/polyphenols/other plat derived chemicals could be bound to it.

Do not vortex or shake DNA solution containing the gDNA. A 260/280nm ratio could tell you something about the purity. Also consider running a few samples on a Bioanalyzer DNA chip. The images from agarose gel separations are hard to quanitfy and the loading dye may obscure the EtBr (or similar) DNA stain.

I have checked the 260/280 ratio of these samples and they range from 1.88 (sample 2) to 1.62 (sample 6). However the 260/230 ratios are somewhat low for all the samples (0.9 to 1.59). Do you think apart from the problems with the carbohydrate contamination (low 260/230), there could be contamination of DNase in RNase enzyme?

 DNA is degraded at the time of process. Please avoid more shearing or vortexing. I think it is not the case of DNase contamination in RNase.

Is this a lyse and boil prep followed by phenol chloroform? Shake the tube and never vortex.  This looks like sheared DNA to me....seems pretty clean though.

Hi ;

İf you are using liquid nitrogen, please only make  dust your leaves with liquid nitrogen and immediatly add extraction buffer. Avoid your sample to get room temperature after you use liquid nitrogen. Because after you disturb the cells with liquid nitrogen your DNA will be effected from DNAses if you dont add extraction buffer to inactive them.

İf  you are using b-mercaptoethanol in your extraction buffer please use it fresh.

And if you collected your leaves before without freezing in liquid nitrogen probably your DNA is degreded at collecting step.

if you are working with a plant which has high levels of polyphenols etc. please add pvp to your extraction buffer.

note: I had the same problem before and I fix my problem with, first advice.

In which solution you are dissolving DNA? What is plant? Because in some plants like wheat if you dissolve DNA in T10E1 buffer then there is a tendency that DNA may get sheared due to triggered DNAse activity or some other reason so, better dissolve DNA either in Sterile de-ionised water or T10 E0.5 bufferor simply TrisCl (10mM) buffer. Purify DNA with Phenol: Chloroform; Isoamyl alcohol (25:24:1) for once and never vortex DNA.

@Arup i had dissolved the DNA in 1x TE buffer and may be i should try to dissolve in Tris-Hcl just to make sure this is not the problem. I am extracting DNA from tree species (Poplar)

Dear Dr. Upendra, can you kindly send me the details protocol as I wish to know what you are doing to remove phenolics/secondary metabolites present in poplar.

 Dear Dr. Arup, i have attached the protocol. Thanks for your help again...

Please try CTAB method and add 1%PVPP (Sigma) while grinding the sample in presence of LiqN2.

Hi Upendra i am sending you the whole procedure for Plant DNA Isolation use this protocol and you will get intact DNA

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

Many thanks Rajput - I will give this protocol a try and will update you all sometime this week of how it went with the modifications.

Pay attention to the DNA contamination, if you do not want the spread of bands. And follow link below

http://artssciences.lamar.edu/_files/documents/chemistry-biochemistry/BL6_DNA_Purification_Analysis_copy.ppsx

http://artssciences.lamar.edu/_files/documents/chemistry-biochemistry/BL6_DNA_Purification_Analysis_copy.ppsx

DNA isolated from tissue sample using the conventional process (read CTAB) will be smeared. Although, the level of smear may vary depending on the process used and how carefully the isolation process was performed.

Can you please mention which ladder used, might give an idea about the size.

Hi! Upendra,

Hope you got out of from your problem by this time and got good DNA. By the way may I know from what leaves are you isolating DNA? In fact I answered many questions about DNA isolation, if you have patience you can search or let me at what stage are you so that I can advise. 

Good Luck!

Hello all, I have finally fixed the problem. I replaced the grinding method (this time in liquid N2) and elution buffer (this time Tris-Hcl) and it worked wonders. I will upload the picture tomorrow. Thanks everyone for all your help..... As promised here it is..

Congratulations!

Glad that solves the problem

Good you have got DNA, hope it works fine.

If you check there is lot of protein junk in the wells. So while separating the phenol, Choloroform, IAA phases do it a bit carefully, do not try to take the last drop of the phase thinking you loose DNA leave the junk there itself then you see only DNA band.

Thanks Yunus, you suggestions actually helped me a lot. Ramadevi - i am not using any phenol/Chloroform based procedure in my protocol. Can you tell me where else i could have making a mistake. Attached is my protocol...

Hi! Let it be any procedure generally plant DNA contain many secondary products, so you should be careful, some times they interfere the PCR. Though you are not using Phenol etc. while extracting the phases take enough care that helps you lot. I believe for PCR you dilute the DNA then there should not be any problem. Otherwise pass through column. By the way what species are you using mentioned some young leaves.

Phenol-Chloroform method is tedious but I always prefer to extract using that method, b'cause we get very pure DNA will not encounter any problems during analysis. . 

Good Luck!