Team:Evry/Notebook/w4
From 2013.igem.org
Line 37: | Line 37: | ||
<p> | <p> | ||
- | <b><u>But :</u> | + | <b><u>But :</u> Aim: Define the number of matrix of optimal DNA to amplify a gene by PCR.</b><br/><br/> |
- | <b><u> | + | <b><u>Information:<br/></u> |
</p> | </p> | ||
<ul> | <ul> | ||
<li> [primer] = --- ng/µl<br/> | <li> [primer] = --- ng/µl<br/> | ||
- | <li> | + | <li> size(primer) = --- nt<br/> |
</ul></b><br/> | </ul></b><br/> | ||
<p> | <p> | ||
- | + | For PCR we chose to use … ng of primer (V=…µL). Same we know size of our primer, we could define the number of primer that we use begin the reaction. | |
- | + | Thanks this expression we define the number of template to take in terms of concentration of sample. | |
+ | <br/><br/> | ||
<b> AJOUTER LA FORMULE </b><br/><br/> | <b> AJOUTER LA FORMULE </b><br/><br/> | ||
- | + | Analysis of results of agarose gel:<br/><br/> | |
<b> AJOUTER LA PHOTO LEGENDEE </b> <br/> | <b> AJOUTER LA PHOTO LEGENDEE </b> <br/> | ||
</p> | </p> | ||
- | <h3> Extraction | + | <h3> Extraction of sequence of wild promoter. </h3> |
<p> | <p> | ||
- | + | Aim: we want have sequences of promoters of genes under the control of transcription factor FUR (Ferric Uptake Regulation). <br/><br/> | |
- | + | We remake PCR samples that failed the last week.<br/> | |
</p> | </p> | ||
<ul> | <ul> | ||
Line 71: | Line 72: | ||
<br/> | <br/> | ||
<p> | <p> | ||
- | + | PCR products are placed on gel and purifiate. After, PCR products are tested in nanodrop.<br/><br/> | |
</p> | </p> | ||
Line 131: | Line 132: | ||
<img src="https://static.igem.org/mediawiki/2013/9/9b/PCR_10-07.png" alt="pcr 10-07" width=250 /> | <img src="https://static.igem.org/mediawiki/2013/9/9b/PCR_10-07.png" alt="pcr 10-07" width=250 /> | ||
- | <h3> | + | <h3>Training a tris-HCl solution (1M) : First solution 100X</h3> |
<p> | <p> | ||
- | + | Aim: Tris-HCl solution will use in a final concentration of 10 mM to resuspend our primers.<br/> | |
</p> | </p> | ||
- | <u> | + | <u>Preparation of the stock solution (1 M, pH 7.5) to a volume of 50 mL:</u><br/> |
<ol> | <ol> | ||
- | <li> | + | <li> Dissolve 4.6g of trisbase in 30 mL of water. |
- | <li> | + | <li> Adjust pH of this solution at 7.5 with concentrated HCl ([c] = --- mol/L // volume ajouter ~ 4 mL). |
- | <li> | + | <li> Adjust volume of the solution at 50 mL with water. |
- | <li> | + | <li> Autoclave the solution. |
</ol> <br/> | </ol> <br/> | ||
<p> | <p> | ||
- | <b><u> | + | <b><u>Note: </u> If the solution is yellow, we should repeat with Tris of best quality.</b> |
<br/><br/> | <br/><br/> | ||
- | <u> | + | <u>Preparation of the solution (10mM, pH 7.5) for a volume of 50 mL.</u><br/> |
- | + | Take 50µL of the stock solution at 1M and add 49.5 mL of water for the dilution. | |
</p> | </p> | ||
- | <h3>Préparation | + | <h3>Préparation of Kanamycine</h3> |
<p> | <p> | ||
- | + | Preparation of 3 solution of 1.5 mL of Kanamycine. | |
</p> | </p> | ||
- | <h2> | + | <h2>Thursday, July 11</h2> |
- | <h3> | + | <h3>Preparation of solution of primer:</h3> |
<ol> | <ol> | ||
- | <li> | + | <li>Centrifuge tubes at 8000 rpm, begin 30 seconds. |
- | <li> | + | <li>Add 250 µL of Tris HCl (10 mM); [Primer] = 100µM |
</ol> | </ol> | ||
<p> | <p> | ||
- | + | We prepare a diluted solution at 5 µM from to the stock solution, for PCR reactions. <br/> | |
- | Dilution | + | Dilution at 1/20: We take 5 µL of the stock solution (100 µM) and we diluate in 95 µL of tris-HCl (10 mM). |
- | + | ||
- | <h3>Transformation | + | <h3>Transformation of BL21:</h3> |
<p> | <p> | ||
- | + | Chemical transformation of BL21 with samples of Cyrille: | |
- | + | ||
<ul> | <ul> | ||
Line 184: | Line 183: | ||
<p> | <p> | ||
- | + | These transformations allow us to do glycerols of these constrctions. | |
</p> | </p> | ||
Line 190: | Line 189: | ||
<ul> | <ul> | ||
- | <li> Fep A (Primers P021 | + | <li> Fep A (Primers P021 and P022) |
- | <li> Fes (Primers P023 | + | <li> Fes (Primers P023 and P024) |
- | <li> sdh C (Primers P025 | + | <li> sdh C (Primers P025 and P026) |
- | <li> ybi L (Primers P027 | + | <li> ybi L (Primers P027 and P028) |
- | <li> ync E (Primers P029 | + | <li> ync E (Primers P029 and P030) |
</ul> | </ul> | ||
Line 200: | Line 199: | ||
<img src="https://static.igem.org/mediawiki/2013/7/75/PCR_11-07.png" alt="pcr 11-07" width=250 /> | <img src="https://static.igem.org/mediawiki/2013/7/75/PCR_11-07.png" alt="pcr 11-07" width=250 /> | ||
- | <h2> | + | <h2>Friday, July 12</h2> |
- | <h3>Vérification | + | <h3>Vérification of transformations</h3> |
<ul> | <ul> | ||
Line 213: | Line 212: | ||
<p> | <p> | ||
- | <b><u> | + | <b><u>note :</u> Note: The negative control was suspect.</b> <br/><br/> |
Les boites de Pétri sont laissés sur la paillasse à température ambiante pour le week-end, les colonies seront repiquée la semaine prochaine. <br/> | Les boites de Pétri sont laissés sur la paillasse à température ambiante pour le week-end, les colonies seront repiquée la semaine prochaine. <br/> | ||
</p> | </p> | ||
- | <h3>Migration | + | <h3>Migration of samples of PCR extraction.</h3> |
<p> | <p> | ||
Line 224: | Line 223: | ||
- | + | The promoter sequence of genes Fep A, Fes, Sdh C, ybi L and ync E were extracted with successfully.<br/> | |
- | + | Samples were purified and, after, tested by nanodrop.<br/> | |
</p> | </p> | ||
Revision as of 15:30, 26 August 2013
4: 8th July - 14th July
Monday, July 8th
We prepared the BL21 and BG1655 strains to be competent.
Tuesday, July 9th
Both strain are not contaminated. |
BL21 are highly competent, BG1655 are little competent. |
Wednesday, July 10th
PCR procedure optimization
But : Aim: Define the number of matrix of optimal DNA to amplify a gene by PCR.
Information:
- [primer] = --- ng/µl
- size(primer) = --- nt
For PCR we chose to use … ng of primer (V=…µL). Same we know size of our primer, we could define the number of primer that we use begin the reaction.
Thanks this expression we define the number of template to take in terms of concentration of sample.
AJOUTER LA FORMULE
Analysis of results of agarose gel:
AJOUTER LA PHOTO LEGENDEE
Extraction of sequence of wild promoter.
Aim: we want have sequences of promoters of genes under the control of transcription factor FUR (Ferric Uptake Regulation).
We remake PCR samples that failed the last week.
- Fec A
- Ent C
- Fec C
- Ace B
PCR products are placed on gel and purifiate. After, PCR products are tested in nanodrop.
Fec A (BG1655) |
Fec A (BL21) |
Ent C (BG1655) |
Ent C (BL21) |
Ace B (BG1655) |
Fec C (BG1655) |
Training a tris-HCl solution (1M) : First solution 100X
Aim: Tris-HCl solution will use in a final concentration of 10 mM to resuspend our primers.
- Dissolve 4.6g of trisbase in 30 mL of water.
- Adjust pH of this solution at 7.5 with concentrated HCl ([c] = --- mol/L // volume ajouter ~ 4 mL).
- Adjust volume of the solution at 50 mL with water.
- Autoclave the solution.
Note: If the solution is yellow, we should repeat with Tris of best quality.
Preparation of the solution (10mM, pH 7.5) for a volume of 50 mL.
Take 50µL of the stock solution at 1M and add 49.5 mL of water for the dilution.
Préparation of Kanamycine
Preparation of 3 solution of 1.5 mL of Kanamycine.
Thursday, July 11
Preparation of solution of primer:
- Centrifuge tubes at 8000 rpm, begin 30 seconds.
- Add 250 µL of Tris HCl (10 mM); [Primer] = 100µM
We prepare a diluted solution at 5 µM from to the stock solution, for PCR reactions.
Dilution at 1/20: We take 5 µL of the stock solution (100 µM) and we diluate in 95 µL of tris-HCl (10 mM).
Transformation of BL21:
Chemical transformation of BL21 with samples of Cyrille:
- Terminateur (T)
- Promoteur (P)
- Plasmide 1K3
- Plasmide 1C3
- sfGFP
These transformations allow us to do glycerols of these constrctions.
PCR sur le génome de E.coli (souche BG1655)
- Fep A (Primers P021 and P022)
- Fes (Primers P023 and P024)
- sdh C (Primers P025 and P026)
- ybi L (Primers P027 and P028)
- ync E (Primers P029 and P030)
Friday, July 12
Vérification of transformations
- Terminateur (T) = OK
- Promoteur (P) = OK
- Plasmide 1K3 = OK
- Plasmide 1C3 = OK
- sfGFP = OK
note : Note: The negative control was suspect.
Les boites de Pétri sont laissés sur la paillasse à température ambiante pour le week-end, les colonies seront repiquée la semaine prochaine.
Migration of samples of PCR extraction.
The promoter sequence of genes Fep A, Fes, Sdh C, ybi L and ync E were extracted with successfully.
Samples were purified and, after, tested by nanodrop.
Fep A (BG1655) |
Fes (BG1655) |
Sdh C (BG1655) |
ybi L (BG1655) |
ync E (BG1655) |
Voir s'il ne faut pas faire le tableau recap des extractions