TITLE: Subcloning colonial ascidians
AUTHORS: Laura Bugada, Simon Blanchoud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We have been subcloning colonial ascidians since some time already, till now - with a lot of success. This protocol has been ruinously used by many of our co-workers.</div></div>

[STEPS]
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Place the cleaned slides to be subclone in the first tray inside a slide rack.
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Take one slide.
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Decide how many additional slides you want to propagate your colony onto.
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Subcloning is typically performed either to recenter systems onto the same slide, to provide more space to large colonies by propagating them onto new slides, or to split a strain into multiple clones.
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Systems from the same clone can be regrouped during subcloning, they will then fuse into one larger clone.
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Wet the new slides with system water, let them dry upwards in the plastic tray.
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Using a sharp razor blade, amputate the systems to be moved.
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If systems need to be split or separated from the rest of the colony, perform the incision first using a gentle vertical slicing movement.
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Detach the systems to be subcloned by performing a horizontal slicing movement on the glass slide. The system should come to rest on the razor blade.
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If needed, slice the excess and/or dirty tunic underneath the subclone to provide a flat attachment surface.
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Take the target slide, dry it, place it horizontally in front of you.
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Transfer the subclone onto the new slide.
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Gently push the subclone from the surface of the razor blade using a wet paintbrush.
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If the subclone is too small to be pushed out of the razor blade, place it on the paintbrush by performing a rolling movement, deposit it on the slide by perform the converse movement.
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Dry the excess water around the subclone.
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Look underneath the subclone, make sure there is no air bubble trapped under it.
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Let the subclone dry vertically in the plastic tray for .
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Using the paintbrush, gently wet the zooids of the subclone. Make sure all tissue gets wet properly
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Dry the excess water and let it dry once more for 5 min.
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Repeat steps 12 and 13
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Let it rest for .
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Take all the subclones slides out of the water, place a maximum of 7 per slide rack, put the whole rack vertically in the quarantine.
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Check all subclones, clean the dead tissue off the slides.
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Move the subcloned colonies to the main system.
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Dry the excess water on the colony using the microfiber tissue.
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Put the subclone upside down on your fingers.
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Hold it gently with your thumb.
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Slice the excess tunic perpendicularly
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Take the slide, enter the water diagonally and place it into the second container vertically.
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If the subclone detaches from the slide, start again from 12.
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Typical issues are a dirty tunic (repeat step 5.3) and dried tissue (repeat step 12).
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Let it rest for  .