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Team:Panama/23 August 2010
From 2010.igem.org
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- 50 ul of TEMED | - 50 ul of TEMED | ||
| - | Then we seal the borders with 2% of agarose, fill with 0,5 TBE x 50 ml and Reveal with | + | Then we seal the borders with 2% of agarose, fill with 0,5 TBE x 50 ml and |
| - | 5 00 ml H2O and 25 ul o Bromure x 20 minutes. | + | |
| + | Reveal with 5 00 ml H2O and 25 ul o Bromure x 20 minutes. | ||
| + | |||
10-15 ul of sample | 10-15 ul of sample | ||
| + | |||
150 V and 17 mamps | 150 V and 17 mamps | ||
| + | |||
12 % | 12 % | ||
| + | |||
- 19,95 ml ABA 10 ml | - 19,95 ml ABA 10 ml | ||
| + | |||
- 3,75 ml TBE 10x 1,87 ml | - 3,75 ml TBE 10x 1,87 ml | ||
| + | |||
- 75 ul APS 37,5 ul | - 75 ul APS 37,5 ul | ||
| + | |||
- 75 ul TE MED 37,5 ul | - 75 ul TE MED 37,5 ul | ||
| + | |||
- 26, 3 ml H2O 13, 15 ul | - 26, 3 ml H2O 13, 15 ul | ||
| Line 33: | Line 42: | ||
- 11 ul H2O | - 11 ul H2O | ||
| + | |||
- 2 ul digested RBS | - 2 ul digested RBS | ||
| + | |||
- 2 ul digested Promotor | - 2 ul digested Promotor | ||
| + | |||
- 2 ul digested plasmid | - 2 ul digested plasmid | ||
| + | |||
- 2 ul T4 10x ligase buffer | - 2 ul T4 10x ligase buffer | ||
| + | |||
- 1 ul T4 ligase | - 1 ul T4 ligase | ||
| + | |||
So to get a final volume of 20 ul, then we incubated this for 2 hours at room temperature | 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 later at 80ºC for 20 minutes to desactivate the enzime. | ||
And good news for everyone, the ligation was done right! | And good news for everyone, the ligation was done right! | ||
| + | |||
so now we have our RBS and promotor in a plasmid =) | so now we have our RBS and promotor in a plasmid =) | ||
| + | |||
{{:Team:Panama/calendar2}} | {{:Team:Panama/calendar2}} | ||
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 =)
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