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Post by lloyd on Nov 4, 2006 18:23:02 GMT -5
Slowly, slowly come my supplies. A friend is going to pick up my 8 litre stainless steel Presto Pressure cooker in Buffalo next week-they are much cheaper in the USA and Amazon ships free in the states. I found some giberellic acid on eBay from a chemical supplier in England; hopefully the real thing. My phytotech supplies are stuck in Customs at Toronto airport and should be here next week too. I found agar and inositol for a good price at Bulkfoods.com. Shoppers' has 3% H202 and 95% isopropyl alcohol. I found a big plastic bin at Canada Tire for a flow hood. I've only been able to get 5% Na0CL bleach at Loblaws. Does anybody know where to get the 10% bleach recommended for sterilization? Can I use the 5% Bleach? Some references mention undiluted 10% bleach, others say to dilute the 10% with water 5-40% by volume before sterilizing. I also made a light system with some odds and ends lying around the house. The furnace room is starting to look like an evil-scientist's laboratory.
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Post by tom on Nov 4, 2006 21:20:31 GMT -5
Hmm a lot of questions here!
About your bin, you are more likely going to build a glove box: a laminar flow hood need a laminar sterile air current, since the use of HEPA filter. In the case your bon will be used with a alcohol burner in an inert air case, we then talk about a 'glove box' in general.
10% bleach: Javel water/chlorox has a concentration of 5% Sodium hypochlorite, a little more or less depending of the brand. But when you read a 10% solution, a 20% solution, it means a solution (volume/volume) of Javel water AND water. So a so called 10% solution would be for example 10 ml of Javel in 90 ml of water for a total of 100 ml (10/100 ml = 10%). 20%? 20 ml in 80 ml of water for, once again, 100 ml. Pure javel seeds soak can be used in a few cases (seeds with very thick/impermeable testa), but it can be risky. I don't usually got higher than a 20-25% solution, even with thought contamination cases. I prefer adding more time to the sterilization, it gives you more flexibility about a living explant/seeds and a dead one. I've never seen any pure 10% javel, and don't see any use to it for this purpose, since you almost never use a 5% javel to its full strenght.
I hope this help.
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Post by lloyd on Nov 5, 2006 0:13:41 GMT -5
Thanks Tom. You're right of course, I'm going to have a glove box. I even have a UVC light source from my electronics supplies to install in it. I'm glad you clarified the bleach concentration for me. Now I'm just waiting not very patiently for the rest of my supplies to come. In the meantime I will play around with my tiny measuring spoons from my aquarium test kits to figure out how to measure milligram quantities of hormones. I have a little scale but the lowest it can measure is 10 mg. which is still too much. Also I want to experiment to see how much vinegar I have to add to get my dehumidifier water from a pH of ~6.5 to around 5.8. Lots of fun to keep me busy over the winter.
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Post by tom on Nov 5, 2006 8:47:15 GMT -5
In the case the weight of your hormone powder (they are on their dry form?) is very small, it would be preferable for you to do a 'master mix', 125 ml of IBA for example at a concentration of 1 mg/ml or something like this, it will be a lot easier to handle, just keep the stock solution in the fridge. Do you know in which product you should mix a cytokinin, in which to mix an auxin? I never remember, but one group need to be in solution first in an acid, the other in a base... If you need this info, i can find it back for you.
A recommandation about your growing area: try to put your flasks into a aquarium with a lid or a similar clear and clean closed area. It will limit a lot the chances that some spores or dusts get into your flasks, or near the lids. In a case of doubts or high contamination (either from your manipulations or the growing area), try to use PPM. Some reports say that the plants grow slower in this stuff, but it's better than having nothing at all! It can be very handy, especially for newcomer which doesn't 'master' the technique yet, or simply for people which dont have all the ideal equipment.
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Post by lloyd on Nov 5, 2006 21:16:14 GMT -5
Thanks again. I'm going to try to do without PPM for now. I've read that it can damage plants and I just want to see if I can achieve practical sterility by good technique. As for the hormones, I ordered them in dry form so I'm going to try to keep them that way until I use them as I think they are more stable in dried form. Between my little scale and my little measuring spoons I should be able to get close to the right concentrations. I'll check on the ideal pH to dissolve hormones, too.
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Post by tom on Nov 12, 2006 9:04:50 GMT -5
I found you an interesting link on PPM: plant-tc.coafes.umn.edu/listserv/1997/log9703/msg00047.htmlI used it once, in my Phalaenopsis heavily contamined stemprop and now, a week later, more than half of the contaminants haven't return, and the explants are still doing great. Auxins stocks (NAA, IBA, IAA, ...) are usually disolved in a few drops of NaOH or KOH (1 M), and once dissolution occured, you can complete with water to your desiredfinal volume, upon wished final concenrtation. Mine is usually o,1 mg/ml. For cytokinins, it is dissolved in HCL (1M). In both case, stocks solutions are kept in the fridge.
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Post by lloyd on Nov 12, 2006 19:00:29 GMT -5
Hi Tom, That is very interesting information about PPM. Based on that, I'm going to order some. Thanks. I wonder if I can get some NaOH and HCL from the hospital where I work. It doesn't seem like I will need much. I have pretty well everything else. My pressure cooker should be coming along soon, too. I also have to get someone to eat the applesauce before I can use the baby food containers (or maybe I can add it to my fruit fly food). Lloyd
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Post by tom on Nov 12, 2006 19:13:34 GMT -5
If you have about 40$, i suggest you the Lydiane Kyte & john Kleyn's book: Plants from test tubes: An introduction to micropropagation, it is a good introduction on every point for TC
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Post by lloyd on Nov 12, 2006 19:19:45 GMT -5
I'll put it on my holiday wish list. Thanks.
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Post by lloyd on Nov 13, 2006 21:34:47 GMT -5
I looked around and it seems that both 2iP and NAA can be dissolved in 1 M NaOH. My son just told me to use Draino (NaOH) granules, 40 gm per litre to make a 1 M. solution. I ordered the smallest amount of PPM (30 ml) from the company. I found the Kyte book for ~$26 on Indigo, less at Amazon.
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Post by lloyd on Nov 16, 2006 22:25:20 GMT -5
Tom (or any TC savants), I was wondering about the hydrogen peroxide which comes as 3% H2O2 in water from Shoppers. The article in the CPN about TC of VFT flower stalks says to use a 3% hydrogen peroxide solution. Now does this mean to use the full strength hydrogen peroxide as it comes out of the bottle (3%H2O2 in water)? Or does it mean make a 3% solution of the stuff that comes out of the bottle-for example take 3cc of the Shoppers hydrogen peroxide and add it to 97cc water? This is similar to the bleach question. Thanks.
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Post by tom on Nov 16, 2006 22:39:42 GMT -5
I think it means to use the peroxide right out of the bottle. Peroxide react pretty quickly and I fear that it might 'lost most of its power' in the water before you even have the time to put the stalk in it. I already used it pure (3%) for orchids and Asclepiads seeds without ill effects. The best thing would be to try a 'test batch' to insure that it is sufficient to sterilize without burning the explants.
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Post by lloyd on Dec 4, 2006 13:42:32 GMT -5
Well my pressure cooker should come this Friday. Then I'll have everything I need (I hope). I even made a super-sensitive scale to measure weights to about 0.1 to 1.0 mg accuracy. I made it out of odds and ends I had in the basement from a website: www.sas.org/tcs/weeklyIssues/2004-11-12/Classics/index.htmlNow that I can weigh mg. quantities, I won't have to use up my 2iP so quickly-I assume it will keep longer as a powder than as a stock solution.
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Post by lloyd on Dec 16, 2006 15:03:39 GMT -5
Well all my supplies are here. I was just wondering about vitamins. I have Inositol. I also could fairly easily mix up some nicotinic acid, pyridoxine and thiamine which seem pretty commonly used in TC. What do people here do about vitamins with CP TC?
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Post by tom on Dec 16, 2006 15:10:15 GMT -5
i dont use them at all, it is surely beneficial, but had good results without them anyway
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