# IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/09/20

(Difference between revisions)
 Revision as of 12:31, 20 September 2012 (view source) (→Result QCM)← Previous diff Revision as of 12:43, 20 September 2012 (view source) (→Result Transformation=)Next diff → Line 6: Line 6: | colspan="2"| | colspan="2"| - =Result Transformation== + ==Result Transformation== - It works weirdly, because there are colonies on positive control, none on negative control, and there is an absence of colonies in the middle of the ligation transformation plates, that can be explain by the platin on Cm on plain LBA plates, and we would have a gradient of antibiotique. + It works weirdly, because there are colonies on positive control, none on negative control, and there is an absence of colonies in the middle of the ligation transformation plates, that can be explain by the platin on Cm on plain LBA plates, and we would have a gradient of antibiotic. So I took 2 colonies from each plate, the ones closer to the hole, and i make them grow in LB+Cm.The culture tube are composed of 5mL of LB + 5µL of Cm. So I took 2 colonies from each plate, the ones closer to the hole, and i make them grow in LB+Cm.The culture tube are composed of 5mL of LB + 5µL of Cm. Line 29: Line 29: * 4°C -$\infty$ * 4°C -$\infty$ -> 25 cycles -> 25 cycles + + * Result of PCR on colonies : + + [[Image:Gel_20_09_PCRcolo.png|thumb|center|500px|Agarose gel after PCR on colonies after transfomrtion of ligation product. Tha bad oare expected : P1003 is about 1000bp in length, and supD+T is a bit more that 200 bp.]] + + Since the PCR is good, the sequence should be good too. We can register 2 new biobricks, and wait for the sequencing results + ==Amp 7*== ==Amp 7*==

## Revision as of 12:43, 20 September 2012

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## Result Transformation

It works weirdly, because there are colonies on positive control, none on negative control, and there is an absence of colonies in the middle of the ligation transformation plates, that can be explain by the platin on Cm on plain LBA plates, and we would have a gradient of antibiotic.

So I took 2 colonies from each plate, the ones closer to the hole, and i make them grow in LB+Cm.The culture tube are composed of 5mL of LB + 5µL of Cm.

I also made a PCR colony with the same colonies.

Here is the content of 1 tube of PCR :

• VR (diluted 10 times from the stock tube) : 2,5µL
• VF2 (diluted 10 times from the stock tube) : 2,5µL
• PCR MM 2X : 25 µL
• water : 20 µL

Here is the PCR pgm :

• 95°C - 15' (to lyse cells too)
• 95°C - 30"
• 50°C - 30"
• 72°C - 40"
• 72°C - 10'
• 4°C -$\infty$

-> 25 cycles

• Result of PCR on colonies :
Agarose gel after PCR on colonies after transfomrtion of ligation product. Tha bad oare expected : P1003 is about 1000bp in length, and supD+T is a bit more that 200 bp.

Since the PCR is good, the sequence should be good too. We can register 2 new biobricks, and wait for the sequencing results

## Amp 7*

My only clone from yesterday grew up in LB+Amp, and I miniprep it and transform it in either NEB turbo or in MG1655 with or without the other plasmid that contain supD

## Result QCM

I get 2 more colonies on Amp and 3 on Tet -> liquid culture.

I also run a gel with the product of Digestion before transformation.

Agarose gel after PCR site directed mutagenesis and Digestion with DpnI. There are 2 samples for each plasmid modified, and W is for water, when the PCR was running without plasmids, only primers. We can see a band for plasmid pSB1A2 and plasmid pSB4K5

The band corresponding to pSB1A1 can explain why we have colonies on Amp plates, but I still don't have colonies on Kan plates, and I have colonies on TetR, it might be contaminants here. Further experiments will make that clear.