Team:Paris Saclay/Notebook/August/11
From 2014.igem.org
(→D - Lemon scent) |
(→Digestion) |
||
Line 19: | Line 19: | ||
-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 | + | 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.
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...