IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/27: Difference between revisions
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| colspan="2"| | | colspan="2"| | ||
* | <br><u>Miniprep</u> | ||
*S200 (Constitutif promoter + standard RBS + tetR) from iGEM08 Vf=60 uL 72.9 ng/ul | |||
*S173 (standard RBS + tetR)from iGEM08 Vf=60 uL 208.3 ng/uL | |||
<br><u>Restriction digest</u> Vf=20 uL | |||
{| | |||
|Sample name||DNA (1ug)||enzyme||Buffer||H2O||BSA | |||
|- | |||
|S173 || 4.8 ||1 S/P ||2(B2)|| 11 ||0.2 | |||
|- | |||
|J23110 (Vf=40uL) || 2 ||2 S/P || 4(B2) ||29.6||0.4 | |||
|- | |||
|B0015 || 7.4||2 X/P ||4 (B2)||24.2||0.4 | |||
|- | |||
|P1003 || 2||1 S/P ||2 (B2)||13.8||0.2 | |||
|- | |||
|C0012 || 6.5||1 S/P ||2 (B2)||9.3||0.2 | |||
|} | |||
<br><u>PCR purification</u> | |||
*P1003 14.8 ng/uL | |||
*S173 17.8 ng/uL | |||
I have made a PCR purification because the fragment cut beetween S and P restriction site will be remove by the column. | |||
<br><u>Electrophoresis</u> | |||
<br><u>Gel extraction</u> | |||
*C0012 11.2 ng/uL | |||
*J23110 well 1 12.4 ng/uL | |||
*J23110 well 2 30.2 ng/uL | |||
<br><u>Overnigth culture</u> | |||
|- | |- | ||
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<font color=red>Aleksandra and Raphaël</font> | <font color=red>Aleksandra and Raphaël</font> | ||
<br><u>Transformation</u> of TOP10 cells (25µl/transformation) with the 07/26 ligations. | <br><u>Transformation</u> of TOP10 cells (25µl/transformation) with the 07/26 ligations: | ||
* TetA(C) + stand.RBS | |||
* RBS/GFP/term + pLux | |||
* weakRBS/LuxR + Pmed | |||
* weakRBS/LuxR + Pstr | |||
* medRBS/LuxR + Pmed | |||
* medkRBS/LuxR + Pstr | |||
* strRBS/LuxR + Pmed | |||
* strRBS/LuxR + Pstr | |||
* LacZ + weak RBS | |||
* LacZ + stand RBS | |||
* LuxI + term | |||
<br>Incubation at 37°C for 21h. | |||
<font color=red>Aleksandra</font> | <br><font color=red>Aleksandra</font> | ||
<br><u>Miniprep</u> | <br><u>Miniprep</u> | ||
<br>4x10ml cultures of term/attC/mRFP, elution with 30µl EB from each column = 120µl DNA in total | <br>4x10ml cultures of term/attC/mRFP, elution with 30µl EB from each column = 120µl DNA in total | ||
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*20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x | *20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x | ||
*40µl attC (miniprep of 07/25) + 1µl SpeI + 1µl PstI + 4.7µl buffer3 10x + 0.45µl BSA 100x | *40µl attC (miniprep of 07/25) + 1µl SpeI + 1µl PstI + 4.7µl buffer3 10x + 0.45µl BSA 100x | ||
<br><font color=red>Théotime</font> | |||
<br><u>DNA purification from the restriction digest reaction using the PCR purification kit</u> | |||
*attC eluted with 30µl of water | |||
<br><u>Making Amp plates</u> | |||
<br>21 plates | |||
<font color=red>Raphaël</font> | |||
<br><u>Gel electrophoresis (1.5% agarose, 50V)</u> | |||
*ladder 1kb | |||
*- | |||
*term/attC/mRFP | |||
*- | |||
*- | |||
*- | |||
[[Image:gel2707.jpg]] | |||
<br><u>Gel purification and nanodrop to quantify DNA</u> | |||
{| | |||
|Sample name||260/280||260/230||ng/µl | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
| attC (PCR purif.kit)|| 1.67|| 1.01||6.2 | |||
|- | |||
|term/attC/mRFP (gel purif.) ||2.01 ||0.05 ||14.4 | |||
|} | |||
<br>We often have very low 260/230 values : probably contamination with the solvents. | |||
<br><u>Ethanol precipitation</u> of the term/attC/mRFP (gel purification) to concentrate the sample. | |||
<br> Following this protocol : [http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids] | |||
<br>60µl ethanol + 2.4µl sodium acetate 3M + 24µl term/attC/mRFP, incubation at -20°C overnight. | |||
<br><font color=red>Aleksandra and Raphaël</font> | |||
<br><u>Overnight cultures</u> | |||
<br> 10mL LB | |||
*pSulib attC - Kan | |||
*pSulib term/attC/mRFP - Kan | |||
*TOP10 pSB1A2 pLux (R0062) - Amp | |||
*TOP10 pSB1A2 RBS/GFP/term (E0240) - Amp | |||
*TOP10 pSB1A2 pLux/GFP (1rst colony on the plate) - Amp | |||
*TOP10 pSB1A2 pLux/GFP (2nd colony on the plate) - Amp | |||
*TOP10 pSB1A2 pLux/GFP (3rd colony on the plate) - Amp | |||
*TOP10 pSB1A2 pLux/GFP (4th colony on the plate) - Amp | |||
Revision as of 06:39, 5 August 2010
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I have made a PCR purification because the fragment cut beetween S and P restriction site will be remove by the column.
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Aleksandra and Raphaël
Raphaël
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