Team:Evry/Notebook/w6

From 2013.igem.org

(Difference between revisions)
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<h2> Monday, July 22nd </h2>
<h2> Monday, July 22nd </h2>
 +
<p>
We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :
We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :
-
- 10 uL of One Taq Buffer 10X
+
</p>
-
- 1 uL of 10 mM dNTPs
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<ul>
-
- 1 uL of each VF2 and VR primers
+
<li>10 uL of One Taq Buffer 10X
-
- 35,5 uL of H2O
+
<li>1 uL of 10 mM dNTPs
-
- 1 uL of the One Taq enzyme
+
<li>1 uL of each VF2 and VR primers
-
- 1 uL of the golden gate products
+
<li>35,5 uL of H2O
 +
<li>1 uL of the One Taq enzyme
 +
<li>1 uL of the golden gate products
 +
</ul>
 +
<p>
We used the following PCR program :
We used the following PCR program :
-
      - 95°C for 3 min
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</p>
-
29 x ( - 95°C for 15 sec
+
-
      - 55°C for 30 sec
+
-
      - 68°C for 2 min  )
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-
      - 68°C for 10 min
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 +
<ol>
 +
<li> 95°C for 3 min
 +
<li> 95°C for 15 sec
 +
<li> 55°C for 30 sec
 +
<li> 68°C for 2 min
 +
<li> 68°C for 10 min
 +
 +
 +
<p>
 +
<b><u>Note:</u> We repeated step 2, 3 and 4 29 times.</b>
 +
</p>
 +
 +
<p>
The gel migration didn't reveal any band. We supposed that it didn't work due to the annealing temperature we used.
The gel migration didn't reveal any band. We supposed that it didn't work due to the annealing temperature we used.
Indeed, with the help of the Tm Calculator website, we found that the optimal annealing temperature for those primers was 53°C.       
Indeed, with the help of the Tm Calculator website, we found that the optimal annealing temperature for those primers was 53°C.       
-
 
+
</p>
<h2> Tuesday, July 23rd </h2>
<h2> Tuesday, July 23rd </h2>
-
In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.
+
<p>
 +
In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.</br>
-
(Mettre photo gel)
+
(Mettre photo gel)</br>
-
According to our results, we can assume that our first PCR was badly realised due to an error of manipulation and not because of the annealing temperature we used.
+
According to our results, we can assume that our first PCR was badly realised due to an error of manipulation and not because of the annealing temperature we used.</br>
In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic.
In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic.
-
 
+
</p>

Revision as of 15:40, 31 July 2013

Iron coli project

Week 6: 22nd July -th 28th July

Monday, July 22nd

We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :

  • 10 uL of One Taq Buffer 10X
  • 1 uL of 10 mM dNTPs
  • 1 uL of each VF2 and VR primers
  • 35,5 uL of H2O
  • 1 uL of the One Taq enzyme
  • 1 uL of the golden gate products

We used the following PCR program :

  1. 95°C for 3 min
  2. 95°C for 15 sec
  3. 55°C for 30 sec
  4. 68°C for 2 min
  5. 68°C for 10 min

    Note: We repeated step 2, 3 and 4 29 times.

    The gel migration didn't reveal any band. We supposed that it didn't work due to the annealing temperature we used. Indeed, with the help of the Tm Calculator website, we found that the optimal annealing temperature for those primers was 53°C.

    Tuesday, July 23rd

    In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.
    (Mettre photo gel)
    According to our results, we can assume that our first PCR was badly realised due to an error of manipulation and not because of the annealing temperature we used.
    In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic.

    Wednesday, July 24th

    Thursday, July 25th

    Friday, July 26th

    Retrieved from "http://2013.igem.org/Team:Evry/Notebook/w6"