Team:Paris Saclay/Notebook/August/11

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(Difference between revisions)
(D - Lemon scent)
(Digestion)
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-Check the replacement of Limonen synthase gene by Apramycin resistant gene in the pJBEI plasmid.
-Check the replacement of Limonen synthase gene by Apramycin resistant gene in the pJBEI plasmid.
-
3 µL plasmid pJBEI
+
1)3 µL plasmid pJBEI (or plasmid pJBEI+ApraR 2)
-
0.5 µ Bgl2
+
0.5 µ BglII
 +
1 µL fast digest buffer
 +
10 µL H2O QSP
 +
 
 +
 
 +
37°C - 1 hour
 +
Migration on agarose gel
 +
 
 +
-Check if the restriction enzymes PacI from Sylvie's and from Alice's stock work.
 +
 
 +
1) 3 µL plasmid pJBEI (or plasmid pJBEI+ApraR 2)
 +
0.5 µ PacI(Alice)
 +
1 µL PacI buffer
 +
10 µL H2O QSP
 +
 
 +
2)3 µL plasmid pJBEI (or plasmid pJBEI+ApreR 2)
 +
0.5 µ PacI(Sylvie)
 +
1 µL PacI buffer
 +
10 µL H2O QSP
 +
 
 +
37°C - 1 hour
 +
Migration on agarose gel
 +
 
 +
Conclusion:
 +
Alice's PacI works because we can see a supplementary band in the pJBEI+ApraR 2 compared to the control but not the Sylvie's.
 +
So, we do have the ApraR gene in our plasmid.
 +
 
 +
-Transformation of E.coli DH5 α by pJBEI+ApraR 2
 +
 
 +
100µL competent bacteria
 +
2 µL pJBEI+Apra 2
 +
 
 +
20'' at 4°C
 +
2''30' at 42°C
 +
2'' at 4°C
 +
 
 +
Then, we add 900µL of lysogeny broth and put it
 +
 
 +
1H at 37°C
 +
 
 +
In parallel, we did a control. (Without adding plasmid)
 +
 
 +
Then, we spread our E.coli on the petri dish containing Solid LB+Apra (1/2000)
 +
100 µL control
 +
100 µL transformated E.coli
 +
200 µL transformated E.coli
 +
100 µL concentrated transformated E.coli
 +
 
 +
Results:
 +
Nothing grew up.
 +
We think that the problem comes from the competent DH5 α that were not really competent...
 +
 
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 11:00, 12 August 2014

Contents

Monday 11th August

Lab work

D - Lemon scent

PCR Clean-up of BBa_K762100

by Sean

PCR prepared on the 7th August.

Clean-up performed with the following protocol.

NB: in the present case we have 90 µl of PCR result and we use 20 µl of elution buffer.

Protocol

Digestion

by Laetitia and Hoang Vu

-Check the replacement of Limonen synthase gene by Apramycin resistant gene in the pJBEI plasmid.

1)3 µL plasmid pJBEI (or plasmid pJBEI+ApraR 2) 0.5 µ BglII 1 µL fast digest buffer 10 µL H2O QSP


37°C - 1 hour Migration on agarose gel

-Check if the restriction enzymes PacI from Sylvie's and from Alice's stock work.

1) 3 µL plasmid pJBEI (or plasmid pJBEI+ApraR 2) 0.5 µ PacI(Alice) 1 µL PacI buffer 10 µL H2O QSP

2)3 µL plasmid pJBEI (or plasmid pJBEI+ApreR 2) 0.5 µ PacI(Sylvie) 1 µL PacI buffer 10 µL H2O QSP

37°C - 1 hour Migration on agarose gel

Conclusion: Alice's PacI works because we can see a supplementary band in the pJBEI+ApraR 2 compared to the control but not the Sylvie's. So, we do have the ApraR gene in our plasmid.

-Transformation of E.coli DH5 α by pJBEI+ApraR 2

100µL competent bacteria 2 µL pJBEI+Apra 2

20 at 4°C 230' at 42°C 2 at 4°C

Then, we add 900µL of lysogeny broth and put it

1H at 37°C

In parallel, we did a control. (Without adding plasmid)

Then, we spread our E.coli on the petri dish containing Solid LB+Apra (1/2000) 100 µL control 100 µL transformated E.coli 200 µL transformated E.coli 100 µL concentrated transformated E.coli

Results: Nothing grew up. We think that the problem comes from the competent DH5 α that were not really competent...


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