Hello (sorry for long text)

I've tried the isolation in three different ways, in the second trial I changed some steps of first trial which are decribed by “ / ” after related step, in the third one I tried completely different procedure. None of the results was good.

I grind whole leaf tissue samples into a fine powder (they seemed like light green flour) in the mortar with the aid of liquid nitrogen and thawing was not allowed.

Here is the first (I) and  second (II) procedure that I followed:

CTAB solution was prepared as;

-       25mL 1M Tris – HCl

-       70mL 5M NaCl

-       10mL 0.5M EDTA

-       5g CTAB

-       Solution was completed to 250mL by adding distilled water.

1.      50 – 100mg plant powder was put into 2mL microcentrifuge tube. / 50-100mg plant powder was put into mortar, and sample was grinded again with aid of liquid nitrogen.

2.      For each sample, 600µL CTAB solution and 8mg PVP were mixed and heated to 60°C. 0.2% β-Mercaptoethanol was added into solution.

3.      CTAB – PVP mixture was added into sample tubes. / 500µL CTAB – PVP mixture was added into mortar, sample was continued to grind, homogenate was put into a 2mL microcentrifuge tube. Mortar and pestle were rinsed with 100µL of CTAB – PVP mixture and this rinse liquid was added to the 2mL microcentrifuge tube.

4.      Sample tubes were incubated at 60°C for 25min, tubes were shaked gently at each 5min. After that tubes were waited at room temperature for 5 – 10min.

5.      450µL 24:1 chloroform : octanol was added to each sample, and samples were centrifuged at 13000rpm for 15min.

6.      Supernatants were transferred into new 2mL microcentrifuge tubes by using slant cut micropipette tips. / 5th and 6th steps were performed twice.

7.      NaCl in ½ volume of supernatant and %95 ethanol in 2 volumes of supernatant were added to samples. Samples were put into freezer (-20°C) for one night.

8.      After overnight incubation, samples were centrifuged at 13000rpm for 10min.

9.      Supernatants were removed. Pellets were washed with 1000µL %75 ethanol and 13000rpm centrifuge for 5min. / This washing step was performed two times.

10.    Supernatants were removed and pellets were dried by using concentrator until they became crisps.

11.    Dried pellets were dissolved in 100µL nuclease – free water.

12.    3µL RNase A was applied to the samples at 37°C for 1 hour.

I have also tried another method (III) with these steps:

1.      0.2g plant powder was put into mortar, and sample was grinded again with aid of liquid nitrogen.

2.      For each sample 500µL squash buffer (2% N – Laurylsarcosine sodium salt, 0.1M Tris – HCl (pH 8.0), 10mM EDTA (pH 8.0)) was added into mortar, sample was continued to grind, homogenate was put into a 2mL microcentrifuge tube. Mortar and pestle were rinsed with 100µL of squash buffer and this rinse liquid was added to the 2mL microcentrifuge tube.

3.      600µL cold phenol was added to the tubes, tubes were turned upside down a few times and incubated on ice for 10min.

4.      Tubes were centrifuged at 13000rpm for 10min.

5.      Upper liquid phases were transferred into new 2mL microcentrifuge tubes by using slant cut micropipette tips.

6.      600µL chloroform was added to the tubes, tubes were mixed well and centrifuged at 13000rpm for 10min.

7.      Upper liquid phases were transferred into new 2mL microcentrifuge tubes by using slant cut micropipette tips.

8.      6th and 7th steps were repeated.

9.      50µL cold 3M NaOAc and 1mL cold 95% ethanol were added into tubes, tubes were turned upside down a few times and put into freezer (-20°C) for one night.

10.    After overnight incubation, samples were centrifuged at 13000rpm for 1min.

11.    Supernatants were removed, pellets were washed by 1mL cold 75% ethanol (13000rpm for 1min) two times.

12.    Supernatants were removed and pellets were dried by using concentrator until they became crisps.

13.    Dried pellets were dissolved in 100µL nuclease – free water.

14.    3µL RNase A was applied to the samples at 37°C for 1 hour.

I am adding the results of the trials.

So in which step I'm doing mistake, I have contaminants and smear, what do you suggest me?