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competent cells: CaCl2 autoclave vs filter sterilisation


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11 replies to this topic

#1 SF_HK

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Posted 08 October 2012 - 12:21 AM

I'm preparing to make competent cells. Is there a reason why some people pefer to fliter sterilise CaCl2 over autoclave? Can CaCl2 be autoclaved?

Thank you

#2 Inbox

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Posted 08 October 2012 - 04:34 AM

I do filter sterilization.
You might want to look @
http://www.protocol-...osts/18679.html

#3 SF_HK

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Posted 08 October 2012 - 06:51 AM

Thank you. Also, to make 0.1M CaCl2, is it necessary to make the dilution from 1m CaCl2 or can I directly make 0.1m CaCl2? I read somewhere that CaCle concentartions must be prepafed from 1M stock solution.

I just simply prepared 0.1M CaCl2 by dissolving 3.67g CaCl2 (dihydrate, FW 147.02) in 250ml water.

#4 Inbox

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Posted 08 October 2012 - 10:01 AM

I just simply prepared 0.1M CaCl2 by dissolving 3.67g CaCl2 (dihydrate, FW 147.02) in 250ml water.


Thats fine.

But how you will make last 0.1M Cacl2 with 15% Glycerol for storing @ - 80oC?

Its quiety handy to make 1M cacl2 , filter it and store @ 4oC. For making 0.1 M Cacl2 just dilute it 10 times by adding to autoclaved distill water.

#5 badguy

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Posted 08 October 2012 - 07:00 PM

i did both types of sterilization and both also works good.
but usually i did filter sterilization because it saves timePosted Image

#6 SF_HK

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Posted 08 October 2012 - 07:41 PM

to make 50ml 0.1M Cacl2 with 15% glycerol, I would add 42.5 ml (0.1M Cacl2), 7.5ml 100% glycerol. This is OK?

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Posted 09 October 2012 - 12:18 AM

to make 50ml 0.1M Cacl2 with 15% glycerol, I would add 42.5 ml (0.1M Cacl2), 7.5ml 100% glycerol. This is OK?


Not ok.

If you add any solution to 0.1 M it will decrease its concentration. You can consider adding cacl2 to 7.5ml 100% glycerol to make it 0.1M cacl2 with 100% glycerol and add 42.5 ml (0.1M Cacl2). But filtering 100% glycerol then for sterilization will be difficult task for u.

Better make 1M cacl2. then dilute accordingly.

#8 SF_HK

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Posted 09 October 2012 - 01:50 AM

Ok,

Lets say I make 1M Cacl2 (filter sterilise).

to make 200ml 0.1M Cacl2 (+15 glycerol)
- 20ml 1M CaCl2 (filter sterilised)
-30ml autoclaved 100% glycerol
-170ml autoclaved water

#9 Inbox

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Posted 09 October 2012 - 02:48 AM

Right.

#10 SF_HK

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Posted 09 October 2012 - 10:16 PM

Thank you. I'm just horrible at mathematics.

#11 mdfenko

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Posted 10 October 2012 - 06:00 AM

Ok,

Lets say I make 1M Cacl2 (filter sterilise).

to make 200ml 0.1M Cacl2 (+15 glycerol)
- 20ml 1M CaCl2 (filter sterilised)
-30ml autoclaved 100% glycerol
-170ml autoclaved water

150ml water
talent does what it can
genius does what it must
i do what i get paid to do

#12 SF_HK

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Posted 10 October 2012 - 08:39 PM

Thank you. Yes I realised the calculation error when I was preparing the solutions.




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