Team:Panama/23 August 2010
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Revision as of 11:52, 26 October 2010
August 23
Today was a productive day.
We prepared 50 ml of Poliacrylamide gel 8% with TBE 0.5M. - 13,3ml ABA (Acrylamide - Bisacrylamide)
- 2,5ml of TBE 10 x
- 34,2ml of H2O
- 50ul of APS 25%
- 50ul of TEMED
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.
We'll use 10-15ul of sample and it is set at 150V and 17 mamps.
12 % poliacrylamide gel
- 19.95ml ABA 10 ml
- 3.75ml TBE 10X 1,87 ml
- 75ulAPS 37,5 ul
- 75ul TEMED 37,5 ul
- 26.3ml H2O 13, 15 ul
After all of this, we finally, started de ligation of RBS, promotor and plasmid.
The ligation mix that we'll use:
So to get a final volume of 20 ul, then we incubated this for 2 hours at room temperature
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.
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