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InvisibleGnuBobo
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Quick cloning procedure overview--all right?
    #4097069 - 04/25/05 10:38 PM (11 years, 7 months ago)

  Thinking about cloning in a little while here, wanted to know if this procedure sounds okay.  Will be inoculating liquid culture--probably 4 just for margin of error. 
    Working in a glovebox, you take the fruitbody.  Then, you split the base with and open the stem up into two halves lengthwise.  Use a sterile scalpel/exacto knife to cut out small pieces of stem material from the inside. 
  Do you need to dunk this in h202?  I think scatmanrav mentioned using a capful of h202 to dunk the tissue in and then toss that into the jar for inoculation.  Is h202 necessary if you're working in a glovebox? 
  And do you use sterilized tweezers to pull out the bits?
 
  Last question: About gloveboxes.  When you're working with tools and surfaces inside a glovebox, and you have the fruitbody you recently picked, how much do you worry about contaminants from the fruitbody getting on your tools, gloves, etc.  I mean, after opening the stem, should you wipe down your glovebox gloves with alcohol, then start to cutting out your tissue pieces?  And is wiping a cup out with rubbing alcohol all right to use as a holder inside the box for sterile tools? 
  I'm a little confused about how "sterile" or clean a glovebox is after misting with iso alcohol and then ousting it and letting it sit.  How much do you need to be concerned about stirring up contaminants inside the glovebox with movements of tools, gloves, etc.
  Thanks very much.  :grin:


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InvisibleHolydiver
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Re: Quick cloning procedure overview--all right? [Re: GnuBobo]
    #4097093 - 04/25/05 10:42 PM (11 years, 7 months ago)

The exterior of the mushroom won't be clean of course, so I like to dunk the entire fruit body in a cup of H202 for 20 seconds or so, before splitting it down the center. You'll see it fizzle awhile, then take it out. Split it down the middle as you described and remove an inner piece of flesh with a utensil that is "as sterile as possible". I find that in a still air glove box, a good wipe on any surface with isopropyl alcohol renders it sterile for a good amount of time. Hope that helps.


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Offlinexburn
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Re: Quick cloning procedure overview--all right? [Re: GnuBobo]
    #4097098 - 04/25/05 10:43 PM (11 years, 7 months ago)

dunking in h202 kills all teh spores and not the mych so why not. I use tweezers. spay the fruit body with iso and then tear


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InvisibleGnuBobo
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Re: Quick cloning procedure overview--all right? [Re: Holydiver]
    #4097104 - 04/25/05 10:45 PM (11 years, 7 months ago)

Thanks.  :thumbup:

I can't afford a flowhood right now so it's gloveboxes for the forseeable future.  I have been wondering just how clean they are for awhile.


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InvisibleHolydiver
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Re: Quick cloning procedure overview--all right? [Re: GnuBobo]
    #4097108 - 04/25/05 10:46 PM (11 years, 7 months ago)

Gloveboxes rule, you don't need a flowhood for this hobby.  They are just a convenient addition to the toy collection down the road.  :cool:


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InvisibleGnuBobo
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Re: Quick cloning procedure overview--all right? [Re: Holydiver]
    #4097122 - 04/25/05 10:48 PM (11 years, 7 months ago)

:rockon:

Cool beans.  :cool:


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