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...

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