User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/21

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 * style="background-color: #EEE"|[[Image:Logo_light_minimal_full_res.jpg|150px]] WiFi coli: A communicolight system
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Changing enzyme... again
 
 * As the previous PCR didn't go well, I decided to use Pfu and to synthesize a new primer, so that, in case that this PCR amplifies nothing, we can use a common polymerase (like rtTh) which, even if it adds an adenine in the 5' extreme, it will not modify the reading frame. I prepared two concentration for each PCR (2), one with a dilution of 1/5 of the ligation with changed RBS PCR product and the other with the normal PCR product's concentration. The following reactions and the tube order are:
 * 1. Positive control. 
 * 2. Negative control. 
 * 3. PCR 1 using Prefix and the primer for the mutation (Reverse) with diluted DNA.
 * 4. PCR 1 using Prefix and the primer for the mutation (Reverse).
 * 5. PCR 2 using primer for the mutation (Forward) and Suffix with diluted DNA. 
 * 6. PCR 2 using primer for the mutation (Forward) and Suffix. 

 --> Mix 1 for 30 μL <--   -H2O > 20.5μl  -Buffer 10x Pfu -> 2.5μl  -MgSO4 > 4μl -dNTP's (20.2mM each dNTP) -> 1μl  -Primer mix (10mM)--> 1μl <br/ > -DNA > 1μl <br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 16.5μl <br/ > -Buffer Pfu --> 2.5μl <br/ > -Pfu-Turbo pol ---> 1.25μl <br/ > <br/ > <br/ > <br/ > - Initialization step: 95°C for 4 min. (only the 1st mix)<br/ >
 * The 40 cycles were programmed as follows:

- Hot start: Stop to add the second mix<br/ >

- Denaturation step: 95°C for 30 seg. <br/ >

- Annealing step: 60°C for 30 seg. <br/ >

- Extension/elongation step: 72°C for 2 min.<br/ >

- Final elongation: 72°C for 10:00 min.<br/ >

- Final hold: 4°C for ∞. <br/ > <br/ >

Purifying plasmid
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 * Today, I also extracted plasmid containing a constitutive promoter that induces the expression of RFP with Roche's kit. The tubes were labelled and stored as Plasmido purif. promotor constit. J23106 Mar 21/06/10 (3-A).


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