TITLE: Slime away: a simple CTAB-based high molecular weight DNA and RNA extraction protocol for "difficult" invertebrates (rev20200518)
AUTHORS: Sergio Vargas, Cüneyt Caglar, Gabi Büttner, Simone Schätzle, Fabian Deister, Gert Woerheide

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocols for the extraction of nucleic acids of recalcitrant non-model invertebrates are lacking. Here we present an extraction protocol for the extraction of DNA and RNA from mucus-rich invertebrate species. We have successfully used the protocol for the extraction of high-molecular-weight DNA from cnidarians and poriferans and to recover high-quality RNA from samples impossible to extract with commercial methods.</div><br/><br/></div>

[STEPS]
?. [Protocol]
Mix the CTAB Buffer in a 15ml falcon tube and preheat it to  before starting the extraction.
60 °C
?. [Protocol]
Add  β-Mercaptoethanol to the preheated CTAB Buffer under the fume-hood directly before you start with the extraction.
100 µl
?. [Protocol]
Cut a small piece of tissue and place it in a 2ml tube. Try to smash the tissue to make cells accessible to the buffer.(Optional)   - Transfer the frozen/preserved tissue to a mortar pre-cooled with liquid nitrogen. Best practice: let the tissue "swim" in the liquid nitrogen until the mortar is dry. The mortar should be kept cold, you can achieve this by placing the mortar in a small cool box with liquid nitrogen in it.   - Grind the tissue until you get a fine powder.   - Using a small spatula, transfer the powder to a 2mL nuclease-free microcentrifuge tube. Best practice: pre-cool the spatula and the tube by placing them in the cool box containing liquid nitrogen this helps to keep the powder dry and cold.
?. [Protocol]
Add  of pre-heated () extraction buffer and  Proteinase K ()  to the tube.
550 µl
60 °C
50 µl
?. [Protocol]
Mix thoroughly until ALL powder OR smashed tissue is in solution. If needed, vortex lightly for ca. . Incubate at least  to overnight at  with moderate mixing (~). In general, if the tissue was ground OR smashed, after two hours the tissue is digested. It is a good practice to manually agitate the digestion approx. every 15 minutes to avoid the formation of clumps of tissue.(Optional) If samples have a high polysaccharide content:  - Add  KOAc ()  to the tubes, mix by inversion and incubate  at  with moderate mixing.  - Centrifuge at max. speed   - Recover () supernatant trying to avoid the transfer of any pellet or viscous precipitate.(Optional) If you expect RNA contamination e.g. in fresh samples:   - Add  RNase A, mix gently by inversion and incubate at  for .
56 °C
33
150 µl
60 °C
600 µl
5 µl
37 °C
?. [Protocol]
Add one volume () of Chloroform to the sample and shake vigorously until the solution turns milky.
600 µl
?. [Protocol]
Centrifuge (full speed) at room temperature for .
?. [Protocol]
Remove the tube carefully from the centrifuge, trying to leave the two phases undisturbed. If the phases mix, centrifuge again. Transfer 2x  of the upper phase to a new 2ml tube. Be careful not to transfer the interphase or traces of Chloroform.
200 µl
?. [Protocol]
Add 1 volume () of isopropanol to the solution containing the DNA. Mix gently by the hand and incubate at room temperature for . You may see tiny bubbles and sometimes white flakes forming.
400 µl
?. [Protocol]
Centrifuge at  for at least  at full speed.
16 °C
?. [Protocol]
Discard supernatant (by pipetting) without disturbing the pellet.
?. [Protocol]
Add  ethanol (  ) to the DNA pellet and centrifuge at maximum speed for  at . The pellet should be visible by now.
1 mL
16 °C
?. [Protocol]
Remove the supernatant carefully (watch for the pellet). If wanted, you can repeat the previous step one more time. If not proceed to 14.
?. [Protocol]
Centrifuge shortly and remove any remaining EtOH with the 10 µl pipette.
?. [Protocol]
Leave the tube open for about  to dry the pellet. You can also check whether the pellet is transparent and proceed to the next step.
?. [Protocol]
Resuspend the DNA in ~ water.  (Optional) Incubate at  in the Thermomixer for .
30 µl
37 °C
?. [Protocol]
Load  of the extracted DNA on a 1% Agarose gel and let the gel run at 40V for  to  hours. If high molecular weight DNA is expected, use the lambda Hind-III marker to assess the quality of the extracts. Quantify the DNA concentration using the NanoDrop: record the concentration, the 260/230 and 260/280 ratios. A good extraction should have 260/230 and 260/280 ratios above 1.8
2 µl
?. The protocol should yield high-quality DNA and RNA. Below is an example using the recalcitrant octocoral Xenia sp. Without KoAc, RNA cannot be recovered on a regular basis. Also, note the quality of the DNA obtained using the protocol. Note: you can use different commercial kits in tandem to isolate either DNA or RNA and purify the extracts further.