Team:MRIIRS FARIDABAD/Lab Notebook

Lab Notebook

January (Week 1-4)

Brainstorming and deciding the direction of the project.

February (Week-5)

• Prior to any wet-lab experiments, the promoter sequences of ADA-regulon were analyzed and their binding sites were taken into consideration.

• Ecocyc was used to extract the desired sequences.

• Snapgene was used for the selection of compatible cloning vectors and their overhangs.

• Primer designing was done with the help of NEB cutter V2.0, NEB Tm calculator and NEB: Double digest finder.

• Primers were ordered.

Week-6

• PCR amplification of promoter of ADA-AlkB gene followed by gel electrophoresis.

• PCR amplification of the reporter gene from EGFP-N1 plasmid followed by gel electrophoresis.

• Gel extraction of amplified sequences.

Week-7

• Sequential restriction digestion of extracted Ada-AlkB promoter using HindIII and EcoRI.

• Sequential restriction digestion of extracted reporter sequence using EcoRI and BamH1.

Week-8

• Ligation of the digested sequences was done using T4-DNA Ligase.

• This ligated DNA was transformed into DH5-alpha E.coli competent cells followed by overnight incubation.

• Colonies were selected for colony PCR.

March (Week-9)

• The transformed colonies were checked again for the desired DNA using plasmid isolation. MDI Mini-prep kit was used for plasmid isolation.

• This was followed by their restriction digestion.

• Gel electrophoresis was conducted.

Week-10

• Gel images were analysed.

• Orders were placed for analytes for conducting assay experiments by artificially spiking water samples.

• Our institute was closed due to COVID 19 and the entire state was under lockdown.