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| Cloning Various ways to create isolated tissue cultues |
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| | #1 (permalink) |
| ExoCannibalist Join Date: Jan 2006
Posts: 5,064
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | 9er Tek Details: A collection of additions
I have quite a bit of people ask me about 9er tek so I thought this thread could be something everyone puts together to add how they tweak little details. Please add any cool ideas that you all use if you use this method. 9er tek is THE BEST CLONING TEK! imho.. This basically is the methods i use with adaptivities to it. It incorporates 9er tek cloning method, Slurry in a Hurry Tek, and some simple things to preserve your clone material for later use in a small fridge. Supplies vacutainers as many 10 cc syringes or 60cc syringes as you want clone material bernzomatic torch room size hepa with ionizer built in large lawn bags 2 exacto knifes osterizer blender 1/2 pint jar 1 quart jar distilled water alcohol swipes foil combination small safe that fits in your fridge 1) First of all, I start this entire cloning process using a shmuvbox as my glovebox. shmuvbox. all sterile work is done in here. http://forums.mycotopia.net/attachme...mg_3672-1-.jpg behold the shmuvbox. best invention of the century! credit to molester. its very portable and you can get a larger liner that will allow more work space. right now you can see 20 quart jars WBS and two BRF jars in this pic. if you zoom in you can see the two hand holes and the eye hole. the more transparent the bag the better. you won't need the eye hole if the bag is transparent. this is such a great invention. i sold my flowhood . i have poured plates, taken clones, made spore syringes, g2g transfers, you name it, it has been done in this thing. portable, effective, cheap! can't go wrong! i use a shmuvbox. it is a HEPA with a big clear trash bag over it, you place all your clean tools, fruits for cloning , and benzo torch to sterilize your blades in this bag. usually i will wipe down every single part of this HEPA with 70-90% rubbing alc. be anal, spray it with disinfectant spray. this thing will pour plates and take clones easily. holmes brand HEPA filters with a small Ionizer built in are a great size. you only need a small room size filter. and it should be around 30$ at walmart. once you put the bag around the filter, tape it down to the table your working on. this will in turn, allow the bag to stay put. now blow your bag up that has all your cooled utensils and sterile equipment in it. IMPORTANT: PUT YOUR HEPA ON THE LOWEST SETTING. YOU WANT SUBTLE AIRFLOW NOT A DRAFT turn on the HEPA and when it blows up, cut two holes in it for your hands. one for each. and if you can't find 100% clear lawn bags for the bag, you can cut eye holes as well. this produces positive air pressure and forces out the bad stuff. its real simple, clones can be taken and it stores nicely. its efficient and takes up little space, and hides easily. 2)Follow the 9er tek in the archives http://archives.mycotopia.net/discus...tml?1004627146 blender usage http://archives.mycotopia.net/discus...tml?1004628678 The only thing different I do is pc two exacto blades for quick use. Pced for 20 mins at 15psi. Allowed to cool for 30=45 minutes. i use two sterile scalpals or exacto knives. exactos are easier to replace the blades and you don't have to reorder from online mushroom supply places. i use one to cut the stem open, the other to cut the tissue out and place in the blender. reason being, i sterilize these with a bernzomatic torch. the bernzo torch has a stand so it stands up. it also has a switch that keeps it going the entire time like a flame lamp only this can melt metal. once the blades are sterile, i cut the stem open. then use the other sterile exacto to cut the inner stem. exactly like 9er tek but in between each flame sterilization, an alcohol swab is used to cool down the blade. you can do this in about 30 mins time with everything and cleanup. i get 1 10cc syringe or even as many as you want to store. i suck up the blended solution into the syringe immediately, rub an alcohol swab on the outside of the vacutainer, then squirt it into a vacutainer. i can sit there and make that solution of clone material into 5-6 viles of vacutainers or even more depending on your volume of water and you stem tissue. one half of a half pint jar filled with distilled water is plenty of clone material. enough for a year if you do this correctly. this is enough for clones for an entire year for me. i store them in the fridge. also distilled water is used for the syringes. you can store them directly in the syringe if that is all you have. i simply just use the metal blade assembly as my temporary lid when im cloning. i lift it up and suck up with the syringe. then place the lid sitting back on top of the jar with solution. not tight just sitting on there. this is almost open air, except it is in the shmuvbox. otherwise, there is no small hole in the lid. its just ghetto. you lift the lid, then suck up water in the syringe. out of 100 syringes...0 contams so its working well. even though a bit ghetto. then you can fill up as many syringes as you want. it may be anal but i always place black electrical tape around my syringes where the lid meets the base. its just an ocd thing i guess. x) Keeping Cultures: Here is a summation of what I do to keep cultures. And it takes up like a small lock box in your fridge that you can keep locked at all times. these will store for 2 years at the least if its clean. i always do a 1 syringe test run in two 1/2pint pf jars and let them grow in 2 days. Speed This is where the speed comes into play. Lets look at it this way, if you have your master sryinges stored in the fridge. All you have to do is inject one syringe into 2 1/2 pint jars. This will grow in a few days with a clone. You then combine this with Tv Casualty's slurry in a hurry tek and you have a clone in a week on grain ready for spawning. Add Tv Casualty's Slurry Tek and.....well there ain't no and. its just fast! http://forums.mycotopia.net/holding-...hree-days.html bernzomatic torch home depot 25$. best 25$ you will EVER spend! guarenteed.. ![]() what this does is preserves your clone. you go back to your original syringes everytime. but if your doing slurry in a hurry to grain. well, then you can use one colonized quart to knock up 12 more quarts for a 2nd generation grain to grain. id stick with 2 generations tops. i had trouble with 3 on a clone with slurry tek. this way, if you make lets say 10-10cc syringes. you get enough clone material for a year or so. i can drag one syringe out for months. its quite fast! the beauty of it, is that it takes up no space for storage in the back of your fridge. also, its very quick and simple. you can make one half pint of mycelia turn into 120 jars in a few days using tvs slurry tek along with this. fastest way to clone! highly suggested!
__________________ Last edited by eatyualive; 10-14-09 at 20:21. |
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| | #4 (permalink) |
| ExoCannibalist Join Date: Jan 2006
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | douse a paper towel with rubbing alcohol and rub down everything. inside, outside, around the plastic part of the filter. place eveything in your bag and then close it. blow the bag up and then you can cut your hand and eye holes. then just spray the lysol in there as if your doing it directly into a glovebox. just to be safe, anytime a hand is taken out, spray some lysol inside your bag through your hand holes before you place it back in. you can never be too anal spraying your lysol. make it a religion! spray through the hand or eye holes you cut. not into the hepa. you can spray down your hepa after you initially clean it with rubbing alc. to be extra careful. as long as your hepa filter is changed every so often. like one time a year your fine. if this is only being used for sterile work, then a filter can last a few years without contamination. if lets say you use this hepa for your room as well. you may want to keep an extra filter on hand that you change out just for sterile work that has less miles on it.
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| | #5 (permalink) |
| Harvester of Sorrow Join Date: Apr 1972
Posts: 1,967
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the ol' bag'n hepa trick ehh? I remember seeing a pic with blue trash bag I think years back of this contraption and wondering if it really works. What kind of contam rates are you getting in there eatters? Can you do agar work in there like pour plates and what not?
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| | #6 (permalink) |
| ExoCannibalist Join Date: Jan 2006
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normal rates upwards of 3%. agar, plates, spore syringes, clones. there are tricks to it though. tape the bag to something when your doing spore work. you want to have little airflow. so fill the bag up with the hepa and tape the top to something above and then turn the airflow off if your working with spores.
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| | #7 (permalink) |
| Alpha Comae Berenices Join Date: Jan 1971
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![]() ![]() ![]() ![]() ![]() ![]() | Those small unites tend to pick up all sorts of funk, so I would definitely recommend just using it for clean work and not using it for the home. If you keep it stored in a trash bag with a spritz of lysol inside its original box it should last forever. I love clones.
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| | #9 (permalink) |
| Former Member Join Date: Aug 2007
Posts: 2,272
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b&d vacutainers tubes http://www.imed.com/shop/detail.cfm/sku/K5124/S/637221 |
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| | #10 (permalink) | ||
| ExoCannibalist Join Date: Jan 2006
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | Quote:
Quote:
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| | #12 (permalink) | |
| Shepherd Join Date: May 2008
Posts: 894
![]() ![]() ![]() ![]() ![]() ![]() | Quote:
eatyualive - I think you're doing awesome things for this forum community and mushroom cultivators in general. You consolidate big ideas into threads. Your work (along with everyone else's) is very appreciated. I hope I can experiment and help as much as you do at some point! | |
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| | #13 (permalink) | |
| ExoCannibalist Join Date: Jan 2006
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | Quote:
the lysol bottle says flammable but it doesn't say anything about the liquid being flammable. not sure on this one. anyone know if lysol is flammable? it hasn't caught fire yet and this has been used about weekly for the last several years. if your worried. don't use it.
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you could also use rubbin alc in a spraybottle but don't spray it when your torch is lit. anyway. what you do is spray the lysol. let it air out. you have all utensils in there. then wipe down your hands with alc. then after the lysol airs out, light your benzo and do your sterile work. you most likely won't take your hands out much, but if so, then rub them down with alc again. and you don't need to spray any lysol. generally foaf will only spray an initial burst of lysol before lets say grain to grain transfers. when cloning foaf will only spray the lysol initially when all utensils are placed in the bag. this airs out a bit, and then all sterile work is done. no sense in risking blowing yourself up. leave that for them terrerists!
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it seems many people are having trouble with the ring around the lids. im not sure why. but here are some close up pics of how i sit it on the lip of the jar. just be very careful when you replace the lid and make sure it fits snug. and you should not have any blender ring issues. this is a 1/2 pint jar and a pint jar to let you know it works fine on any size jar as long as it has a small mouth lid.
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