Team:Panama/23 August 2010

August 23

Today was a productive day.

We prepared 50 ml of Poliacrylamide gel 8% with 0.5 x TBE.

- 13,3ml ABA (Acrylamide - Bisacrylamide)

- 2,5ml of TBE 10 x

- 34,2ml of H2O

- 50ul of APS 25%

- 50ul of TEMED

Then we have to seal the edgez with 2% of agarose. 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.

10-15ul of sample

150V and 17 mamps

12 % poliacrylamide gel

- 19,95 ml ABA 10 ml

- 3,75 ml TBE 10x 1,87 ml

- 75 ul APS 37,5 ul

- 75 ul TE MED 37,5 ul

- 26, 3 ml H2O 13, 15 ul

After all of these, we finally, started de ligation of RBS, promotor and plasmid.

The ligation mix that we use was:

- 11 ul H2O

- 2 ul digested RBS

- 2 ul digested Promotor

- 2 ul digested plasmid

- 2 ul T4 10x ligase buffer

- 1 ul T4 ligase

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.