User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/17

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Starting with the punctual mutation on luciferase
      -H2O > 38.2μl  -Buffer Pfx -> 5μl  -MgSO4 > 1μl  -dNTP's (10μM)---> 1.5μl  -Primer mix (10μM) --> 1.5μl  -DNA (PCR ligation product) ---> 2μl  -Pfx -> 0.8μl  <br/ > -H2O > 38.2μl <br/ > -Buffer Pfx -> 5μl <br/ > -MgSO4 > 1μl <br/ > -dNTP's (10μM)---> 1.5μl <br/ > -Primer mix 1 (10μM) > 1.5μl <br/ > -Primer mix 2 (10μM) > 1.5μl <br/ > -DNA (PCR ligation product) ---> NONE<br/ > -Pfx -> 0.8μl<br/ > <br/ > <br/ > -H2O > 38.2μl <br/ > -Buffer Pfx -> 5μl <br/ > -MgSO4 > 1μl <br/ > -dNTP's (10μM)---> 1.5μl <br/ > -Primer mix (10μM) --> 1.5μl <br/ > -DNA (plasmid 6) -> 2μl <br/ > -Pfx -> 0.8μl<br/ > <br/ > <br/ > --> Mix 1 for 30 μL <-- <br/ > <br/ > -H2O > 11.5μl <br/ > -Buffer 3.3 -> 6μl <br/ > -Mg(OAc)2 -> 3μl <br/ > -dNTP's --> 2.5μl <br/ > -Primer mix 1 --> 2.5μl <br/ > -Primer mix 2 --> 2.5μl <br/ > -DNA > NONE <br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 10.5μl <br/ > -Buffer 3.3 --> 9μl <br/ > -rtTh polymerase --> 0.5μl <br/ > <br/ > <br/ > <br/ > --> Mix 1 for 30 μL <-- <br/ > <br/ > -H2O > 13μl <br/ > -Buffer 3.3 -> 6μl <br/ > -Mg(OAc)2 -> 3μl <br/ > -dNTP's --> 2.5μl <br/ > -Primer mix 1 --> 2.5μl <br/ > -DNA > 2μl <br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 10.5μl <br/ > -Buffer 3.3 --> 9μl <br/ > -rtTh polymerase --> 0.5μl <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > --> Mix 1 for 30 μL <-- <br/ > <br/ > -H2O > 13μl <br/ > -Buffer 3.3 -> 6μl <br/ > -Mg(OAc)2 -> 3μl <br/ > -dNTP's --> 2.5μl <br/ > -Primer mix > 2.5μl <br/ > -DNA > 2μl of a 1/5 dilution <br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 10.5μl <br/ > -Buffer 3.3 --> 9μl <br/ > -rtTh polymerase --> 0.5μl <br/ > <br/ > <br/ > <br/ > <br/ > - Initialization step: 94°C for 4 min. (only the 1st mix)<br/ >
 * As referred | previously, the mutation on luciferase requires the amplification by PCR of the two fragments so that, when we use only the prefix and suffix, the mutation is there. I did the following reactions for the PCR using both Pfx and rtTh as a control.
 * 1. Pfx with Prefix and the primer for the mutation (Reverse) with DNA dilution of 1/5.<br/ >
 * 2. Pfx with Prefix and the primer for the mutation (Reverse).<br/ >
 * 5. Pfx with primer for the mutation (Forward) and Suffix with DNA dilution of 1/5.<br/ >
 * 6. Pfx with primer for the mutation (Forward) and Suffix.<br/ >
 * 3. Pfx Negative control using both, Prefix and primer for the mutation (Reverse) and primer for the mutation (Forward) and Suffix.
 * 4. Pfx Positive Control using Prefix and Suffix.
 * 7. rtTh Negative Control using both, Prefix and primer for the mutation (Reverse) and primer for the mutation (Forward) and Suffix.
 * 8. rtTh Positive Control using Prefix and Suffix as primers and plasmid 6.
 * 9. rtTh with Prefix and the primer for the mutation (Reverse) with DNA dilution of 1/5.<br/ >
 * 10. rtTh with Prefix and the primer for the mutation (Reverse).<br/ >
 * 11. rtTh with primer for the mutation (Forward) and Suffix with DNA dilution of 1/5.<br/ >
 * 12. rtTh with primer for the mutation (Forward) and Suffix.<br/ >
 * The 35 cycles were programed as follows:

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

- Denaturation step: 94°C for 15 seg. <br/ >

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

- Extension/elongation step: 68°C for 1 min.<br/ >

- Final elongation: 72°C for 5:00 min.<br/ >

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


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