User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/08

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More work...


  • I started the day by plating the transformed strains that did grow well. Still, results weren't as expected :(


  • 1. rtTh Negative Control using Prefix and Suffix.


  • 2. rtTh Positive Control using Prefix and Suffix as primers and plasmid 18 with 7+6+5.


  • 3. rtTh with Kanamicin primer and the primer for the mutation of the rbs with DNA dilution of 1/10.


--> Mix 1 for 30 μL <--

-H2O ------------> 8μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 5μl
-Primer 1 ------> 3μl
-Primer 2 ------> 3μl
-DNA ------------> 2μl


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

-H2O ---------------------> 10.5μl
-Buffer 3.3 --------------> 9μl
-rtTh polymerase ------> 0.5μl


  • The 30 cycles were programed as follows:



- Initialization step: 94°C for 4:30 min.

- Hot start: Stop to add the second mix 2.

- Denaturation step: 94°C for 45 seg.

- Annealing step: 60°C for 45 seg.

- Extension/elongation step: 72°C for 3:50 min.

- Final elongation: 72°C for 10:00 min.

- Final hold: 4°C for ∞.