From 2014.igem.org

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...

Back to the calendar