Team:Panama/23 August 2010

From 2010.igem.org

Revision as of 18:58, 14 October 2010

August 23

Today was a productive day.

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

- 13,3 ml ABA (Arilamid - Bisacrilamid)

- 2,5 ml of TBE 10 x

- 34,2 ml of H2O

- 50 ul of APS 25%

- 50 ul of TEMED Then we seal the borders with 2% of agarose, fill with 0,5 TBE x 50 ml and

Reveal with 5 00 ml H2O and 25 ul o Bromure x 20 minutes.

10-15 ul of sample

150 V and 17 mamps

12 %

- 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 =)