Team:IISER Bhopal/Notebook

iGEM IISER Bhopal

Notebook

MAY

First Week of May
  • Beginning of lab work.
  • SOB,SOC, TE, TB, TAE prepared
  • LB media and LB agar plates prepared
  • Competent cell preparation of E . coli (DH5a) carried out but failed

Second Week of May
  • Comp cell preparation of E. coli (DH5a) attempted thrice and failed.

Third Week of May
  • iGEM kit arrived.
  • Comp cell preparation of E. coli (DH5a) successful and competency checked.
  • PUC-18 plasmid isolation and stock preparation.

Fourth Week of May
  • AFP, CPN-10, CPN-60 transformation carried out individually.
  • Plasmid isolation of the above transformations carried out.

Fifth Week of May
  • Primer resuspension followed by primer stock preparation carried out.

JUNE

First Week of June

Second Week of June

Third Week of June
  • Gradient PCR-2 for Cpn-60 carried out but failed.
  • IPTG + X-Gal plates prepared.
  • Ligation of PUC-18+AFP carried out successfully.
  • Ligation of PUC-18+CPN-10 carried out but failed.
  • SOEing PCR of AFP-CPN-10+CPN-60 carried out but failed.

Fourth Week of June

JULY

First Week of July

Second Week of July

Third Week of July
  • Colony PCR of P. frigidicola carried out but failed.
  • Digestion of Cpn-10 in PUC-18 carried out successfully.
  • Plasmid isolation for Cpn-10+PUC-18 carried out.
  • PCR carried out to confirm if the plasmids are correct.

Fourth Week of July
  • Digestion of Cpn-60+psBIC3 carried out successfully.
  • Sequential digestion of Cpn-60 in PUC-18 carried out.
  • Colony PCR for TF in P. haloplanktis in PUC-18.
  • Plasmid isolation for Cpn10 in pUC18.
  • And for Cpn60 in pSB1C3.
  • Checking plasmids for Cpn10 in pUC18 - by PCR.

Fifth Week of July

AUGUST

First Week of August
  • Characterization of Cpn-60.

Fifth Week of August
  • Sequencing of PUC-18+Cpn-10+Cpn-60 carried out.

SEPTEMBER

First Week of September
  • Amplification of pET 28a carried out but failed.
  • Amplification of P. frigidicola TF carried out.

Second Week of September
  • Transformation of iGEM part- stress sensor pSB1C3 carried out in Rosetta and DH5a selection in Rosetta was not successful (reason: Rosetta is inherently resistant to chloramphenicol).
  • Plasmid extraction from a transformed DH5a colony was carried out.
  • Overlapping PCR carried out to replace Lac promoter with Ptrc promoter at different annealing temperatures(56 ℃, 57 ℃, 58 ℃, 59 ℃).
  • From the above PCR amplification, two fragments were extracted(1085bp & 2018bp) and were further amplified.
  • Overlap extension PCR with the amplified fragments was carried out.
  • Digestion of the products of overlap extension PCR and pET 28a was carried out(with same restriction enzyme).
  • Ligation and cloning of digested product was carried out.

OCTOBER

Growth Curves