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OfflineMrBlueshrooms
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Registered: 07/11/21
Posts: 332
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Mr.B's MM tek for crossing strains * 1
    #28054807 - 11/16/22 04:45 PM (1 year, 2 months ago)

Mr.B's Mutual Maceration Tek

  Maceration is a technique used de-dikaryotize a mated strain. This can be done chopping or grinding.
  So let's macerate our target strains together, then let the monokaryotic cells sit in close proximity en masse. Use clones or ms plates.

Equipment

  I use an X shaped stir bar that is ment to stir a litre. It is flat bottomed as opposed to having a pivot ring. More grinding power.
  The jar is 250ml tall. 3/4 full.
  Here is a pic with 2 strains settled together:


And you need a stir plate.

Stages

  Seperation if using agar donors for parents, if using LI or LC, skip this stage.  After an initial chop, cultures are left on medium low speed to seperate the cultures from the agar. Satellite cultures will make many new cells at this stage. New cell growth is initially monokaryotic. (Watling)
  2 days.

  Maceration grind the cultures to free the monokaryotic cells.
  1 hour.

  Fermentation lets the cells settle and sit in close proximity for mating. 2 days.

  Preperation gently resuspend the mycellium. At this stage you can squirt to grain, which is what I do, or you can plate it and select for vigorous iso's.

Which brings up an interesting point. Even if you are working from clones, the genetic recombination will lead to many varieties.

 
  Excecution. Here are my notes from my recent TW x Nat cross, I have used it on cube cube crosses too. Other times, speeds Will work I'm sure.

  Day 1 Set stir plate so vortex reached the bar. (5 for me).
  60 minutes.
  Set to 2.5, just pulling the vortex into the liquid. Leave for 2 days.

  Day 3 Maceration time.  Power 5 again for 50 minutes. Turn off stir plate.

  Day 5 inoculation time! Resuspend cells by stirring at low power for a few.

There are a few ways people create their crosses, I present this as an additional tool.


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Offlinemykophil
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Registered: 10/31/19
Posts: 130
Last seen: 8 hours, 16 minutes
Re: Mr.B's MM tek for crossing strains [Re: MrBlueshrooms]
    #28068270 - 11/25/22 05:26 AM (1 year, 2 months ago)

do you use 2 monokaryons at the start?
if not, how do you know, if the 2 different strains or species did actually really bind or clap with each other? or can you only tell later after the suspected cross is sequenced?
sounds really cool thanks for the write-up of this tek !


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