I did succesfull DNA extraction for diatoms with the soil DNA extraction kit from QIAGEN (Protokoll for maximum yields). Good luck! Anja

You can improve the isolation by performing cell lysis by macerating the cells with liquid nitrogen followed by a pre-treatment with nuclear lysis buffer. The DNA extraction can be performed with phenol:chloroform:isoamylalcohol (Trizol or Roth) with further CIA extraction. This worked for my experiments in chromatin isolation from Chlamydomonas. If you want any other detail, you may find some other info in my thesis (http://opus.kobv.de/ubp/volltexte/2011/5390/pdf/winck_fv_diss.pdf)

Here is a method we use for Chlorella

Harvest Cultures:

1. Aliquot 2 x 100 ml cultures into 4 x 50 ml Falcon tubes. Each tube should contain 40-45 ml culture.

2. Centrifuge cells at 5000 rpm for 5 min at room temp. Check that most cells are pelleted. It is possible to remove all of the supernatant to this stage and freeze the pellet in liquid nitrogen. Store tubes in -80oC freezer.

3. If not storing the 50 ml tube, perform the following steps: Remove all but ~5 ml culture medium from two of the tubes and all of the culture medium from the other two. Resuspend two pellets in 5 ml culture medium (leaving two tubes with 5 ml in each).

4. Transfer 1.25 ml culture medium to 8 tared sterile 2 ml screw cap tubes. Label tubes with strain and date.

5. Centrifuge in microfuge for 1 min at 10,000 rpm. Check to see that cells are pelleted.

6. Decant supernatant and re-weigh tubes to determine fresh weight in each tube.

7. Freeze tubes liquid nitrogen and store at -80oC.

Nucleospin II Plant Kit

1. Add 500 ul of PL1 to 100 mg of flash frozen Chlorella cells. Resuspend on ice, add to an autoclaved 2 ml screwtop tube containing 300 mg .4-.6 micron acid washed beads.

2. Vortex at 3000 rpm for 15 minutes. Tape samples to vortex head, do not use the larger vortex holder as the cell walls will not be opened well.

3. To reduce excess foam quick spin samples at 14,500 rpm for 1 minute. Remove liquid from tube and transfer to a fresh 1.5 ml eppendorf tube.

4. Add 10 ul of RNAse A and place in a 37 oC water bath or incubator for 10 minutes.

5. Remove liquid from samples and place into the Clarification Column (violet ring). Centrifuge for 2 minutes at ≥ 11, 000 g. Remove flow through and place into fresh 2 ml tube.

6. Add 450 ul PC buffer to Flow Through and pipet 5 x times to mix well.

7. Add 700 ul of PC buffer/Flow Through Combination to DNA Binding Column (green ring). Centrifuge for 1 minute at ≥ 11, 000 g. Discard flow through. Add any remaining PC buffer/Flow Through to the column and repeat centrifugation conditions. Discard flow through.

8. Wash the membrane with 400 ul of PW1. Centrifuge for 1 minute at ≥ 11, 000 g and discard flow through.

9. Wash the membrane with 700 ul of PW2. Centrifuge for 1 minute at ≥ 11, 000 g and discard flow through.

10. Wash the membrane with 200 ul of PW2. Centrifuge for 2 minute at ≥ 11, 000 g and discard flow through. This will dry the membrane thoroughly.

11. Place column in fresh collection tube. Add 70 oC 25 ul of PE (elution buffer) directly to the middle of the binding column. Allow to sit at room temperature for 5 minutes. Centrifuge for 1 minute at ≥ 11, 000 g.

12. Add 70 oC 25 ul of PE (elution buffer) directly to the middle of the binding column. Allow to sit at room temperature for 5 minutes. Centrifuge for 1 minute at ≥ 11, 000 g.

13. Analyze sample on the nanodrop. 260/280 ratio should be 1.8-1.9, 260/230 ratio should be 1.9-2.0.

14. Label tube with sample name, concentration, date. Store in -20 oC until needed.

Hi! Look... for Chlorella at least, we use here at lab a "classic" DNA extraction. Here... first, collect cells by culture sample centrifugation (4000rpm x 5mins m/l) in a small eppendorf tube.We need a pellet of the size of a lentil. Then, resuspend cells with TEN for to wash culture medium. After that, with distilled water, lysis buffer, some small glass spheres and a small plunger, Lysis buffer is SDS, NaCl, EDTA and pH tris. Then, separate proteins and DNA with a mix of phenol:(chloroform:isoamylalcohol) and centrifugation. Finally you must precipitate DNA with cold ethanol to -20ºC for 2 hr at least. It works for Chlorella, Tetraselmis, Phaeodactylum, etc... =) I hope it works for you!

You can improve the isolation by performing cell lysis by macerating the cells with liquid nitrogen followed by a pre-treatment with CTAB buffer, cells lysis at 60°C for 30'.

Are you sure that the quantity of cells is appropiate? maybe your cultures are quite poor... (¿?) Chlorella has cell wall... here it works with classic method... is rare that it doesn't work with dry ice...

try again CTAB method for extraction of DNA from pure culture of Chlorella, we extract good quality and quantity DNA from this microalgae by CTAB method

We succeeded after pre-macerating the microalgal cells featuring a tough cell wall with a cellulase preparation usually employed for obtaining of higher plant chloroplasts.

Thank you all for your answers, I'll try again using the CTAB buffer and increasing the cell density of my cultures, as Pamela said I think this could be my main problem. I'll alsotry to use liquid nitrogen as I have read that it has better results than using dry ice.

Thanks!

lots of different kits out there, with the majority using similar approaches described above. If you want a paper that explores DNA extraction from Microalgae and evaluates DNA yield and quality I would recommend Lucy Eland who published a paper entitled "Evaluation of DNA extraction methods for freshwater eukaryotic microalgae." in Water research. 07/2012; 46(16):5355-64. DOI:10.1016/j.watres.2012.07.023

This method is a low cost and high quality genomic DNA preparation from Gram-positive bacteria, yeast and fungi.

1. Grow 5ml overnight culture. Pellet the culture at 14000xg in a microcentrifuge for 3 min at room temperature.

2. Discard the supernatant and resuspend the pellet in 1.5ml water. Pellet and remove the supernatant.

3. Resuspend the pellet in 200ul lysis buffer (2% Triton X-100), 1% SDS, 100mM NaCl, 100mM Tris-HCL, pH 8.0, 1mM EDTA).

4. Transfer to a screw cap microcentrifuge tube (Enzymax EZC200SN: $55/500 tubes or other screw cap tubes).

5. Add 200ul phenol.

6. Add 300ul glass beads or zirconium beads. Use bead beater or vortex mixer to vortex continuously for 4-5 min. Keep the tube on ice and centrifuge at full speed in microcentrifuge for 5 min at 40C.

7. Carefully transfer the upper phase (~100-150ul) to a new microcentrifuge tube avoiding phenol carryover from the lower layer and beads.

8. Add 1ml 100% ethanol and wait 4 min at room temperature. Centrifuge at full speed for 5 min. Remove the supernatant.

9. Wash the pellet with 70% ethanol and centrifuge as above.

10. Air dry the pellet and resuspend in 100ul ddH2O+RNase (95 μL dH2O + 5 μl10mg/ml DNase free RNase A).

11. (Option for high quality DNA) Remove RNase A and cleanup the DNA by using DNA cleanup kit (EZC200, $55/50 preps).

Note: You can also buy Enzymax bulk DNA Mini spin columns (EZC101, $36/100 columns) or DNA Tini Spin columns (EZC106. $33/50 columns) and we will provide the solution recipes for you to make you own solutions in the kits. Please contact [email protected] if you need the solution recipes for DNA miniprep, DNA gel extraction and PCR cleanup kits.

I am agree with Pamela Labbé-Ibáñezi used the similar protocol but at the place of SDS i used sarkosyl. i isolated the genomic DNA successfully from Chlaydomonas, Scenedesmus, Chlorella and also from some cyanobacteria.

I agree with Pamela. CTAB is one of the apropriate classical methods for plant DNA extraction. Just try increase microalgae sample concentration or if it still don't, try Qiagen DNeasy plant kit.

I have done DNA extraction for Chlorella and Scenedesmus before using Nucleospin Plant II kit from Macherey Nagel. Normally, I'd use fresh algal cultures and each time, it was a success. U should try!

Thank you. I finally succeded by using the genejet plant dna extraction kit from thermo scientific along with glass beads. Thank you all for your nice suggestions!!