Team:Heidelberg/Templates/DelH week1


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29-04 - 05-05-13

Generation of Plasmid Backbones

Transformation of Biobricks

Part of the registry Plate of 2012 Well Resistance
pSB4K5 (insert=J04450) 1 5G Kanamycin
pSB6A1 (insert=J04450) 1 1K Ampicillin
lacZ (I732019) in pSB1AK3 4 12G Kanamycin, Ampicillin
AraC (I0500) in pSB2K3 1 14N Kanamycin
pSB1C3 (insert=J04450) 1 3A Chloramphenicol
  • Added 10 µl H2O to each well
  • Incubated for 10 min at RT
  • Thawed 5x chemical competent E.coli Top10
  • 3 µl of plasmid DNA were added
  • Incubated for 10 min on ice
  • Heat shock for 40 s at 42.2°C
  • Incubated for 10 min on ice
  • Added 500 µl LB Medium
  • Incubated at 37°C for 40 min
  • Centrifuged 120 at 5,000 rpm, supernatant discarded
  • Pellet resuspended in remaining medium
  • Plated on plates with the corresponding antibiotics (as shown in the table above, section: resistances)
  • Incubated for 2 days at RT
  • One colony was picked from each plate and incubated over night at 37°C in LB medium with the antibiotic listed above


All 5 parts from the Registry Distribution 2012 were successfully transformed in E.coli Top10 except for the one containing the AraC promotor.

Amplification of DelH Fragments

Design of Primers

Identifier Order date Note Sequence
DN01:DelH_f1_PacI_fw 03-05-2013 Amplification of DelH F1, with RBS and adding PacI restriction site tttt ttaattaa tcacacaggaaagtactag ATGGACCGTGGCCGCCTGC GCCAAATCG
DN02:DelH_f1_SalI_rev 03-05-2013 Amplification of DelH F1 until SalI restriction site tttt GTCGACCAACACCTGTGCCTGC
DN03:DelH_f2_SalI_fw 03-05-2013 Amplification of DelH F2 starting at SalI restriction site tttt GTCGACTGGATGGAGCCTGGTGAAAG
DN04:DelH_f2_KpnI_rev 03-05-2013 Amplification of DelH F2, adding KpnII restriction site tttt ggtacc TCAGTCCAGCGCGTACTCCAG
DN05:AraCbb_KpnI_fw 03-05-2013 Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding KpnI site tttt ggtacc aaagaggagaaatactagatgaccatg
DN08:AraCbb_PacI_rev 03-05-2013 Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding PacI site tttt ttaattaa gctagcccaaaaaaacgggtatg