Team:Panama/23 August 2010

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{{:Team:Panama/Header2}}
==='''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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The ligation mix that we'll use:
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We prepared 50 ml of Poliacrilamid gel 8% with 0.5 x TBE.
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[[Image:Cuadro23agosto.jpg|250px|thumb|left|alt text]]
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- 13,3 ml ABA (Arilamid - Bisacrilamid)
 
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- 2,5  ml of TBE 10 x
 
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- 34,2 ml of H2O
 
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- 50  ul of APS 25%
 
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- 50 ul of TEMED
 
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Then we seal the borders with 2% of agarose, fill with 0,5 TBE x 50 ml and
 
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Reveal with 5 00 ml H2O and 25 ul o Bromure x 20 minutes.
 
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10-15 ul of sample
 
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150 V and 17 mamps
 
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12 %
 
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- 19,95 ml ABA      10    ml
 
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- 3,75  ml TBE 10x  1,87  ml
 
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- 75    ul APS      37,5  ul
 
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- 75    ul TE MED  37,5  ul
 
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- 26, 3 ml H2O      13, 15 ul
 
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After all of these, we finally, started de ligation of RBS, promotor and plasmid.
 
-
 
-
The ligation mix that we use was:
 
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- 11 ul H2O
 
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- 2  ul digested RBS
 
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- 2  ul digested Promotor
 
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- 2  ul digested plasmid
 
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- 2  ul T4 10x ligase buffer
 
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- 1  ul T4 ligase
 
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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.
 
-
 
-
And good news for everyone, the ligation was done right!
 
-
 
-
so now we have our RBS and promotor in a plasmid =)
 
 +
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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