User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/14

From OpenWetWare
Jump to navigationJump to search
Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

PCR day


  • Today, I wanted to do two PCRs: one in order to change the RBS of the luciferase as described in the parts registry, and another to conduct a punctual mutation for the emission espectrum of luciferase.


  • The first assay failed, as I missed the hot start step. The reaction was done as follows for 6.5 reactions:


--> Mix 1 for 30 μL <--

-H2O ------------> 74.75μl
-Buffer 3.3 -----> 39μl
-Mg(OAc)2 -----> 19.5μl
-dNTP's ---------> 16.5μl
-Forward -------> 16.5μl
-Reverse -------> 16.5μl
-DNA -----------> (2μl for the tubes that required it)


--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)

-H2O ---------------------> 68.25μl
-Buffer 3.3 --------------> 58.5μl
-rtTh polymerase -----> 3.25μl


  • The 30 cycles were programed as follows:

- Initialization step: 95°C for 5 min. (only the 1st mix)
- Hot start: Stop to add the second mix
- Denaturation step: 95°C for 45 seg.
- Annealing step: 60°C for 45 seg.
- Extension/elongation step: 72°C for 1:50 min.
- Final elongation: 72°C for 5:00 min.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Mariana_Ruiz_Velasco_L./Notebook/IGEM_2010/Wet_lab_journal/2010/06/14&oldid=423925"