Team:Panama/23 August 2010

From 2010.igem.org

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==='''August 23'''===
==='''August 23'''===
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After all of this, we finally, started the ligation of RBS, promoter and plasmid.
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Today was a productive day.
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'''We prepared 50 ml of Poliacrylamide gel 8% with TBE 0.5M.'''
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- 13,3ml ABA (Acrylamide - Bisacrylamide)
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- 2,5ml of TBE 10 x
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- 34,2ml of H2O
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- 50ul of APS 25%
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- 50ul of TEMED
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We have to seal the edges with 2% of agarose so the poliacrylamide doesn't leak out. Then we have to fill the system with 50ml of TBE 0.5M  and it is stained with 500ml of H2O and 25ul of Ethydium Bromide for 20 minutes.
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We'll use 10-15ul of sample and it is set at 150V and 17 mamps.
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'''12 % poliacrylamide gel'''
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- 19.95ml ABA      10    ml
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- 3.75ml TBE 10X  1,87  ml
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- 75ulAPS      37,5  ul
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- 75ul TEMED  37,5  ul
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- 26.3ml H2O      13, 15 ul
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After all of this, we finally, started de ligation of RBS, promotor and plasmid.
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The ligation mix that we'll use:  
The ligation mix that we'll use:  
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So to get a final volume of 20 ul, then we incubated this for 2 hours at room temperature
 
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and later at 80ºC for 20 minutes to desactivate the enzime.
 
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And good news for everyone, the ligation was done right!
 
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so now we have our RBS and promotor in a plasmid.
 
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So to get a final volume of 20ul, then we incubated this for 2 hours at room temperature and later at 80ºC for 20 minutes to deactivate the enzyme.
 +
And good news for everyone, the ligation was done right! So now we have our RBS and promotor in a plasmid.
{{:Team:Panama/calendar2}}
{{:Team:Panama/calendar2}}

Latest revision as of 21:48, 26 October 2010

iGEM Panama

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August 23

After all of this, we finally, started the ligation of RBS, promoter and plasmid.

The ligation mix that we'll use:

alt text









So to get a final volume of 20ul, then we incubated this for 2 hours at room temperature and later at 80ºC for 20 minutes to deactivate the enzyme. And good news for everyone, the ligation was done right! So now we have our RBS and promotor in a plasmid.

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