I am sharing this procedure which is little bit time consuming, but useful, you may try, Harvest algae cells, grind with liquid nitrogen in mortar pestle till it get  fine  powder as like talc powder, add around 1.5 ml extraction buffer, and then add 0.7 ml of suspension buffer, add SDS (10%), keep it on WB at 65oC for 20-30 min, centrifuge,  supernatant,   add 0.8ml Ammonium Acetate, keep it at4oC  for 1h , centrifuge, add iso-propanol  in supernatant, and keep it in -20oC for overnight, centrifuge, discard supernatant wait till alcohol got evaporated, dissolve DNA pelette with TE or water and you may use further. Best Luck

I am not sure how much you should expect. It of course depends on the species and the amount of sample. I do not think the problem is the method; i would rather work on its optimization

what type of microalgae are you working with? I work a lot with diatoms and even for different diatom species you will get different yields when using the same method!  Most column based kits have not yielded good amounts of diatom gDNA and I often use an  "old-fashioned method" grinding in liquid N2 followed by lysis at elevated temperature and then phenol/chloroform extractions. The only "kit like" product that has worked for me is a modified version of this prodcut"www.promega.de/~/media/files/resources/protcards/wizard genomic dna purification kit quick protocol.pdf". I include a glass bead lysis after adding the nuclei lysis solution.

So, you are doing 18S PCR prior to send for sequencing? I have a method that works with just 1.0mL of algal culture with a considerably low biomass concentration. Check my papers to see the method/reference, This one use glass beads.  Your DNA extraction yield should be enough for PCR, maybe you just need to clean it up / concentrate?

What kind of sequencing do you intend to do?

For Sanger sequencing your amount is more than enough, so I assume you want to do NGS.

I trialled 5 different extraction methods with 5 different algal species, using a bead better to lyse the cells. Using the Chelex method, I got the highest amount of DNA, although fairly diluted. With the 5 Prime Archive Pure DNA extraction kit I got the highest concentrations on average, and the second highest amount of DNA. The Bioline, and two different QIAGEN methods gave me variable results.

Looking at the total DNA, most of these extractions resulted in enough DNA for doing NGS.

I've never done CTAB on algae, only on fish, but the method usually works extremely well. How much algae are you using for the extraction? How are you lysing the cells?

I think if you optimise your input of algae and your cell lysis, you can increase your concentration and DNA yield.

It is a hard question because 'microalgae' is too broad to be treated with a single method. However, 30 ng, would make me happy in many cases because for some reason (physiological status, quality of cell wall, mucilage, etc), with some species obtaining DNA is more difficult than others. If you have sufficient sample you can try to concentrate at the end. Good luck.

Thanks to all. I read all the information. It is very useful. 

I am working with Chlorophytes and Cyanophytes from different hot spring water.  

I am trying with 16s and rbc L markers. 

According to Raju information, how much amount of biomass is needed?

If 30ng is enough mean, I could not see the clear band after electrophoresis. 

Do not rely 100% on the electrophoresis. I use the Sybr family of fluorochromes, and sometimes the gel results are altered somewhat. Some researchers are faithful to EtBr and say that never fails. You see, in the laboratory of molecular biology one sways between theory, myth and individual experience, a rather confusing world! My recommendation is to persevere, always.

1) Dear All, 

 This my protocol, please check it and instruct me where I want to get make changes.........

500mg of microalgal biomass washed with ddH2O at 10000 g for 2 min remove the supernatant.

2) The pellet washed with 4% Triton 100X at 10000 g for 1min and remove the supernatant

3) Add 400 µl of (pre-warmed to 60°C) CTAB extraction buffer, Add 40 µl of PVP (1%) + 10 µl of Proteinase K (20mg/ml) on the pellet vortex for 5 min at maximum speed and incubate at 60°C for 1h by using thermo shaker, adjusted to low speed (300rpm) during incubation

4) After incubation, spin the mixture at 12000 g for 5 min to spin down cell debris. Transfer the supernatant to clean tubes

5) Add 400 µl chloroform and mix tube by inversion for 2 min (use rotator at 20 rpm speed)

6) Centrifuge tubes at 14000 g for 10 min at 4oC and wait 1 min at 4oC to let layers settle down. Transfer upper-aqueous phase to a new microfuge tube

7) Add 50 μl of 7.5 M ammonium acetate to the same tube followed by 500 μl of ice cold absolute ethanol (96 %)

8) Invert the tube slowly several times to precipitate the DNA and the tubes can be placed for 1 hr at 20°C after the addition of ethanol to precipitate the DNA

9) Spin the solution for 15 min at 14000 g for 15 min at 4oC and remove supernatant carefully (now you pellet DNA, next step is to wash it out)

10) Add 750 µl ice-cold EtOH (70%) and rotate it for 2 min (use rotator at 20 rpm speed).

11) Centrifuge the tube at 14000 g for 1 min at room temperature to form the washed pellet

12) Discard supernatant and let the DNA dry by leaving tube open in sterile cabinet for appx 10 min and then add 50-100 µl sterile T10E1 or DNAase free ddH2O

13) Use nanodrop for spectrophotometric quantification at Abs 260/280.

My sample concentration varies from 100mg to 500mg biomass, and tried with  SDS and Mercaptoethanol also precipetation I have used isopropanol (300ul).

By following this I got 6 to 21 ng/ul of DNA and some of the samples I got negative values like -6.96 and -15.43 by today

Please suggest me some more........ Thanks in advance

The only problem I can see is the cell lysis step.

Whenever I didn't lyse the cells well enough, I got very small amounts of DNA or RNA.

What do you mean by this: "40 µl of PVP (1%)"

Are these beads?

I am not using any beads only vortex the cells.

40 µl of (1%) PVP  was added before vortex.

There are a few other steps that you do differently to our standard CTAB protocol.

But considering you are working with cells that live in a hot environment, I suspect that just vortexing and then heating for 1 hour is simply not enough to rupture the cells.

As I said, when I was optimising my extractions, I found that I got bad yields when I failed to completely lyse the cells.

I would look into either using a bead beater, or grinding your cells with liquid nitrogen. Alternatively, there are little plastic "pestles" that fit into 1.5 mL tubes, which can be used to grind the cell within a tube. However, they don't work as well as the two above-mentioned methods: http://www.thomassci.com/Equipment/Homogenizers/_/72628210-F46B-44F2-AF74-6DB77AC18825?q=Homogenizing%20Pestles

For physical grinding, use a bead dispenser to load your vials and plates with your desired amount of beads.

Here is a bead dispenser that I work with. Also using a large number of samples. LabTIE also has a large variety of Bead dispensers, fitting tubes, vials, 96-well 384-well etc.

https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers

https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers

Dear Gurudeeban, I do not know if the concentration obtained is total or per microliter. If it's for microliter, it's more than enough, I would not worry.

Another aspect that I notice is that you are using CTAB, does that mean that you work with mucilaginous organisms? If this is the case, I would recommend you to try InstaGene (BioRad) and go straight to PCR.

Best regards,

Rosaluz

Hi Gabrielle, I've only tried it with desmids and dinophyta. If I do not get enough material at first, re-amplify on the first amplicon. Both quantity and quality are good.

regards