I am using the following protocol for silica-gel dried leaf samples with a lot of success in orchids (and other plants). You can have a try (see the steps below) or Qiagen Plant Mini Kit should also work fine.

1. The day before extraction add polyvinylpyrrolidone (M.W. 40,000 Fisher Cat#: BP431-500) to the CTAB buffer and put it in a water bath at 65 °C to dissolve PVP. If β-mercaptoethanol (Fisher Cat#: BP176-100) is needed add this at the same time. CTAB buffer with PVP needs to be used within 2-3 days, store capped. For 5 ml CTAB add 0.015 g PVP and 10 ul β-mercaptoethanol. Modify accordingly if different volume of extraction buffer is needed.

2. Wipe a leaf with 70% ethanol to remove any bacterial and fungal contaminants

3. Grind 0.01 g of silica-dried leaf in a 1.5 ml eppendorf tube with a pellet pestle over liquid nitrogen. Add a small amount of sand using a small steel spatula, to aid grinding

4. Add 800 μl of the pre-warmed (65 °C) CTAB buffer onto the powder and grind a bit more. Incubate for 60 min at 65 οC. Mix by inverting every 5 min

5. Centrifuge at 13000 rpm for 5 min. Following centrifugation, you should have two layers: top: aqueous phase, bottom: debris. Go on to the next step quickly so the phases do not remix

6. Using a pipette carefully transfer the upper aqueous phase to a new 1.5 ml eppendorf tube. Avoid removing any material from the debris

7. Add an equal volume of chloroform:iso-amyl alcohol (24:1) (in the fume hood) and mix well (e.g. 5 min in a rotary shaker or converting the tubes manually for 5 min) to obtain an emulsion (excessive shaking will degrade the DNA).

8. Centrifuge at 13000 rpm for 10 min. Following centrifugation, you should have three layers: top: aqueous phase, middle: debris and proteins, bottom: chloroform. Go on to the next step quickly so the phases do not remix

9. Using a pipette carefully transfer the upper aqueous phase to a new 1.5 ml eppendorf tube. Avoid removing any material from the interface

10. Repeat the chloroform extraction (steps 7-9)

11. Add 2 volumes (≈1000 μl) of ice cold 96% ethanol and 0.08 volume (of the total volume ≈85 μl) of 7.5 M ammonium-acetate

12. Mix well and let sit in freezer for 60 min to overnight. Longer times will tend to yield more DNA, but also more contaminants. Longer times of precipitation is needed for old herbarium material

13. Centrifuge at 14000 rpm for 12 min to pellet

14. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA

15. Add 200μl of cold 70% Ethanol and mix. Leave it stand for 5 minutes

16. Centrifuge for 4 min at 7000 rpm

17. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA

18. Repeat 70% ethanol wash

19. Dry the pellet: Invert samples on a Kim-wipe and let stand with open lid for 15-25 min on lab-bench. Avoid over-drying, suspend the pellet when it is damp

20. Resuspend samples with 50 μl of TE buffer. Gently flick the tube to resuspend the pellet and leave on RT for an hour, then place the tubes into refrigerator for at least overnight. On the following day you have your DNA-extract ready to use.

If you are gentle enough to your DNA during the above extraction steps, you can have a very high quality and clean extract. I used such extracts for AFLP with normal performance. Alternatively, you can always use Qiagen, which is much faster.

Cheers

you might ask Qiagen if they have any kits for this purpose.

Have you tried to add PVP in your extraction buffer to remove the mucus?

I have extracted DNA from fresh tissues, but this will be a dehydrated tissue. Moreover, for AFLP the quality of DNA must be very high. Thats why I was thinking about some specific protocols.

Use 2% CTAB DNA extraction protocol, with Chloroform and Phenol. It has worked for DNA extraction from sweet potato and AFLP amplifications

I have extracted DNA from silica gel preserved leaf samples of orchids (genus Jumellea) using Qiagen DNEasy Plant mini kit adding PVP in the extraction buffer. I then used DNA to amplify microsatellite loci (~200bp) and chloroplast sequences (~800bp). According to measurements with the Nanodrop and electrophoretic gels, DNA seems to be pure and good quality.

I am using the following protocol for silica-gel dried leaf samples with a lot of success in orchids (and other plants). You can have a try (see the steps below) or Qiagen Plant Mini Kit should also work fine.

1. The day before extraction add polyvinylpyrrolidone (M.W. 40,000 Fisher Cat#: BP431-500) to the CTAB buffer and put it in a water bath at 65 °C to dissolve PVP. If β-mercaptoethanol (Fisher Cat#: BP176-100) is needed add this at the same time. CTAB buffer with PVP needs to be used within 2-3 days, store capped. For 5 ml CTAB add 0.015 g PVP and 10 ul β-mercaptoethanol. Modify accordingly if different volume of extraction buffer is needed.

2. Wipe a leaf with 70% ethanol to remove any bacterial and fungal contaminants

3. Grind 0.01 g of silica-dried leaf in a 1.5 ml eppendorf tube with a pellet pestle over liquid nitrogen. Add a small amount of sand using a small steel spatula, to aid grinding

4. Add 800 μl of the pre-warmed (65 °C) CTAB buffer onto the powder and grind a bit more. Incubate for 60 min at 65 οC. Mix by inverting every 5 min

5. Centrifuge at 13000 rpm for 5 min. Following centrifugation, you should have two layers: top: aqueous phase, bottom: debris. Go on to the next step quickly so the phases do not remix

6. Using a pipette carefully transfer the upper aqueous phase to a new 1.5 ml eppendorf tube. Avoid removing any material from the debris

7. Add an equal volume of chloroform:iso-amyl alcohol (24:1) (in the fume hood) and mix well (e.g. 5 min in a rotary shaker or converting the tubes manually for 5 min) to obtain an emulsion (excessive shaking will degrade the DNA).

8. Centrifuge at 13000 rpm for 10 min. Following centrifugation, you should have three layers: top: aqueous phase, middle: debris and proteins, bottom: chloroform. Go on to the next step quickly so the phases do not remix

9. Using a pipette carefully transfer the upper aqueous phase to a new 1.5 ml eppendorf tube. Avoid removing any material from the interface

10. Repeat the chloroform extraction (steps 7-9)

11. Add 2 volumes (≈1000 μl) of ice cold 96% ethanol and 0.08 volume (of the total volume ≈85 μl) of 7.5 M ammonium-acetate

12. Mix well and let sit in freezer for 60 min to overnight. Longer times will tend to yield more DNA, but also more contaminants. Longer times of precipitation is needed for old herbarium material

13. Centrifuge at 14000 rpm for 12 min to pellet

14. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA

15. Add 200μl of cold 70% Ethanol and mix. Leave it stand for 5 minutes

16. Centrifuge for 4 min at 7000 rpm

17. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA

18. Repeat 70% ethanol wash

19. Dry the pellet: Invert samples on a Kim-wipe and let stand with open lid for 15-25 min on lab-bench. Avoid over-drying, suspend the pellet when it is damp

20. Resuspend samples with 50 μl of TE buffer. Gently flick the tube to resuspend the pellet and leave on RT for an hour, then place the tubes into refrigerator for at least overnight. On the following day you have your DNA-extract ready to use.

If you are gentle enough to your DNA during the above extraction steps, you can have a very high quality and clean extract. I used such extracts for AFLP with normal performance. Alternatively, you can always use Qiagen, which is much faster.

Cheers

I used DNA extracted from dry infected wheat leaves samples and it worked well for SSRs and some sequencing.

See details at:

A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping.

https://www.researchgate.net/publication/51507379_A_rapid_genotyping_method_for_an_obligate_fungal_pathogen_Puccinia_striiformis_f.sp._tritici_based_on_DNA_extraction_from_infected_leaf_and_Multiplex_PCR_genotyping?ev=prf_pub

Article A rapid genotyping method for an obligate fungal pathogen, P...

If mucilaginous is the problem for quality DNA extraction, drying up the leaf samples may help. You may to be careful not to get the sample contaminated. Also, phenol extraction step may also help to get quality dna even if you use fresh leaf samples. I would suggest you to try this step.

I think any CTAB-based protocol will work fine, but maybe you should modify some steps depending on the species' characteristics. I have been doing DNA extractions from carnivorous plants, which are very difficult to work with because the high amount of mucilage and other problems but Doyle and Doyle (1987) protocol with modifications by Lodhi et al. (1994) has worked fine for my samples.

LODHI, M.A.; GUANG-NING, Y.; WEEDEN, N.F. & REISCH, B.I. A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis. Plant Molecular Biology Reporter 12(1): 6–13. 1994.

On the other hand, for AFLP analysis, you must be careful regarding the quality of the DNA instead of the quantity.

Good luck!

Hi Paromik,

I would recommend the PowerPlant Pro DNA Isolation kit from MO BIO Laboratories but following the protocol for dried plant tissue. You should use only 5-10 mg of the dried leaf.

I would be happy to send you a free sample of the kit for you to try. Let me know.

@Shi Liu made a good suggestion about adding PVP to Qiagen kits.

There is actually a paper that explores modifying a couple of different plant DNA extraction kits in this manner - albeit from wood tissue, not orchids. See attached.

Search for Gawenda et al and phalaenopsis. She used a modified ctab protocol to get aflp quality dna! Br

Collect the fresh tender leaf, then immediately subjected at freezing point after 25minutes applied to lyophilization. Place the leaf samples in a polystyrene plastic container or some type of container able to hold liquid nitrogen. Add liquid nitrogen to quick-freeze samples. Once frozen, do not allow samples to thaw until freeze-dried. Leaf samples may be stored at –80°C until ready to be lyophilized. After this processed sample to be used for the common procedure. Refer These Links

http://www.science.siu.edu./plant-biology/faculty/nickrent/Plant.DNA.html

http://www.gramene.org/newsletters/rice_genetics/rgn14/v14p164.html

http://www.mobot.org/MOBOT/research/applied_research/methods.shtml

I'm wondering how long everyones plant tissue samples have been stored in silica? I need to extract DNA from museum samples of ficus leaves stored in silica, some of which are 9 years old....any thoughts?

Dear you should use Farmentas DNA Isolation kit.... for your leaf samples of Orchids.