IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/12: Difference between revisions
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==8/12== | ==8/12== | ||
* Transformed ligation mixture | |||
<u>Ethanol</u> | |||
Got plasmid PACYC Duet-pdc-adhb in strain DH10b and strain BL21 from Nielsen's lab to use for ethanol production | |||
*Plated both in preparation to transform plasmid into BL21 Strain. | |||
<u>Transformed ligation mixture</u> | |||
##Ligation mixture | ##Ligation mixture | ||
##- Control | ##- Control | ||
##+ Control (ACC Plasmid) | ##+ Control (ACC Plasmid) | ||
PCR Amplification of ACC | PCR Amplification of ACC parts <br style="clear:both;"/><br> | ||
First, the dry primers were made into 100 μM master stocks. 1 μL was added per .1 nm. For example, a 31.2 nm sample had 312 μL added. This master stock was then diluted into working stocks of 5 μM. This was done by adding 1 μL of master mix into 19 μL H2O. This was done for all eight primers , four forward and four reverse. | |||
The ACC plasmid was then diluted from 54.2 μg/μL to about 1 μg/μL through a 1/50 dilution. | |||
< | PCR mix for all four reactions (accA, accB, accC, and accD) | ||
: PCR reaction | |||
{| {{table}} border="1" align=center | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | | |||
|- | |||
|| 2x GoTaq Master Mix || 12.5 | |||
|- | |||
|| Forward Primer (working stock)|| 1.0 | |||
|- | |||
|| Reverse Primer (working stock) || 1.0 | |||
|- | |||
|| Template DNA (Diluted) || 1.0 | |||
|- | |||
|| H<sub>2</sub>O || 9.5 | |||
|- | |||
|| Total || 25.0 | |||
|- | |||
|| Initial denature step of 95 °C for 2 min | |||
|- | |||
|| PCR cycle: 95 °C for 30 sec -> 45 °C for 45 sec -> 73 °C for 80 sec | |||
|- | |||
|| Final annealing step at 73 °C for 5 min | |||
|- | |||
Revision as of 11:04, 15 August 2014
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8/12Ethanol Got plasmid PACYC Duet-pdc-adhb in strain DH10b and strain BL21 from Nielsen's lab to use for ethanol production
PCR Amplification of ACC parts
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