I've been trying to isolate DNA from Dunaliella microalgae using the plant DNAzol protocol without been successful yet. The yield is quite low with too poor quality DNA. I observed that after centrifuging the microalgae + DNAzol, the supernatant where the DNA is contained is still green colored, and after adding the Ethanol 99.9% for precipitation, I obtain a very big green pellet which is difficult to eliminate after several washes. Do you have any suggestion to get rid of the microalgae chemical components and improve this protocol or another straightforward protocol for this issue?
Thanks in advance
CTAB is a good way to go
https://www.ijsr.net/archive/v3i9/U0VQMTQxMDU%3D.pdf
http://wordpress.clarku.edu/debrobertson/laboratory-protocols/dna-extraction-from-marine-algae-and-seagrasses/
Have you ever tried phenol-chlorophorm extraction after CTAB? This type of extraction will eliminate most of the polar and apolar substances, leaving almost only DNA and RNA (if done correctly). It can be very usefull for particularly hard samples!
See this protocol by monsanto:
Weigh out 6g of processed tissue into a 50ml conical tube appropriate for centrifugation. Note: For unprocessed tissue, weighing m ay occur prior to processing as long as entire processed sample is transferred to the conical tube.
For each 6g sample add 25ml of a solution consisting of 24.25ml, pre-warmed (55°C) CTAB extraction buffer, 0.5ml 2-mercaptoethanol (2-ME), and 0.25 ml of 10mg/ml proteinase K for a final concentration of 2% (2-ME) and 100µg/ml (proteinase K).
Incubate the tube for 60 minutes at 55°C. Cool the tube briefly on bench (10 minutes)
Add 20ml of phenol:chloroform:isoamyl alcohol (PCI, 25:24:1). Cap the tube and mix vigorously by vortex or inversion.
Centrifuge for 10 minutes at 13,000×g and 20-25°C to separate the aqueous and organic phases. Transfer the upper aqueous phase to a clean 50ml conical tube.
Repeat extraction two times for a total of three extractions (steps 4-5).
Transfer upper aqueous phase to a new tube and add approximately 2/3 volume of -20°C isopropanol and gently invert the tube several times to mix.
To precipitate the DNA place the tubes at -20°C for at least 30 minutes and up to three days.
To pellet the DNA centrifuge the tubes at approximately 13,000×g for 20 minutes at 4°C.
Redissolve the pellet in 4 ml of TE pH 8.0. Transfer to a 13ml Sarstedt tube and add approximately 40µl of 10mg/ml RNase, then incubate at 37°C for 30 minutes.
To extract the DNA add 4ml of chloroform:isoamyl alcohol (CIA, 24:1). Centrifuge for 10 minutes at approximately 13,000×g at room temperature. Transfer the upper aqueous phase to a clean Sarstedt tube.
Repeat step 11 then add half volume of 7.5M ammonium acetate, gently mix by inversion/pipette and add 2 volumes of 100% ethanol. Mix by inversion/pipette and place at -20°C for 30 minutes to overnight.
Centrifuge at 13,000×g for 20 minutes at 4°C to pellet the DNA.
chloroform-phenol method can help you to solve problem. The green parte containing clorophil sure will bind to another macro-molecules such as DNA
Scott Newman's miniprep might work for you: https://www.chlamycollection.org/methods/minipreps-of-dna-from-chlamydomonas-cultures/
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