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

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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
-DNA ------------------------------------> 1μl

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

-H2O ---------------------> 16.5μl
-Buffer Pfu --------------> 2.5μl
-Pfu-Turbo pol -----------> 1.25μl

  • The 40 cycles were programmed as follows:

- Initialization step: 95°C for 4 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 95°C for 30 seg.

- Annealing step: 60°C for 30 seg.

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

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

- Final hold: 4°C for ∞.

Purifying plasmid

  • 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).