IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/04: Difference between revisions
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==Preparing another RBS-TesA and LacI Ligation== | ==Preparing another RBS-TesA and LacI Ligation== | ||
* Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone). | * Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone). | ||
: TesA+RBS insert with LacI+backbone | |||
{| {{table}} border="1" align=center <!-- Digestion table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | RBS-TesA (μL) | |||
| bgcolor=#cfcfcf | LacI (μL) | |||
|- | |||
|| DNA || 15.0|| 15.0 | |||
|- | |||
|| 10X fast Digest Buffer|| 2.0 || 2.0 | |||
|- | |||
|| PstI || 1.0 || 1.0 | |||
|- | |||
|| XbaI|| 1.0 || .0 | |||
|- | |||
|| SpeI || 0.0 || 1.0 | |||
|- | |||
|| H<sub>2</sub>O || 1.0 || 1.0 | |||
|- | |||
|| 37°C for 1 hour | |||
|- | |||
|} | |||
*Both samples used in the Clean and Concentrate kit | |||
*Treat the LacI with Antarctic Phsophotase. | *Treat the LacI with Antarctic Phsophotase. | ||
: Antarctic Phsophotase Treatment | |||
{| {{table}} border="1" align=center <!-- Digestion table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | | |||
|- | |||
|| LacI DNA || 10.0 | |||
|- | |||
|| Antarctic Phosphotase|| 1.0 | |||
|- | |||
|| Phosphotase Buffer || 2.0 | |||
|- | |||
|| H<sub>2</sub>O || 7.0 | |||
|- | |||
|| 37°C for 1 hour then 65°C for 5 min | |||
|- | |||
|} | |||
*The RBS-TesA and LacI DNA samples were quantified: | *The RBS-TesA and LacI DNA samples were quantified: | ||
RBS-TesA: 39.157 ng/μL | *RBS-TesA: 39.157 ng/μL | ||
LacI: 62.579 ng/μL | *LacI: 62.579 ng/μL | ||
*Overnight ligation reactions were conducted for the test ligation and the negative control. | *Overnight ligation reactions were conducted for the test ligation and the negative control. | ||
: TesA+RBS insert with LacI+backbone | |||
{| {{table}} border="1" align=center <!-- Digestion table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | 1 (μL) | |||
| bgcolor=#cfcfcf | Control (μL) | |||
|- | |||
|| TesA+RBS || 1.25 || 0.0 | |||
|- | |||
|| LacI || 1.0 || 1.0 | |||
|- | |||
|| Ligation Buffer || 1.0 || 1.0 | |||
|- | |||
|| Ligase || 1.0 || 1.0 | |||
|- | |||
|| H<sub>2</sub>O || 5.75 || 7.0 | |||
|- | |||
|| 16°C overnight | |||
|- | |||
|} | |||
<u>Ethanol:</u> | |||
*Started seeds of 3 seperate colonies of ethanol plasmid | |||
**50 ml LB w/ Chloramphenicol + picked colony | |||
**Cells didn't grow fast enough to add IPTG to start protein growth, so cells were left overnight to grow more | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 20:33, 6 August 2014
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Preparing another RBS-TesA and LacI Ligation
Ethanol:
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