Genetic lab diary

1st week: 2.07.13 – 5.07.13

Preparation of competent cells for chemical-based transfection. Failure.

2nd week: 8.07.13 – 12.07.13

Succeed preparation of competent cells for chemical-based transfection. Verification with iGEM’s transformation kit. Preparation of competent cells for electroporation. Failure.

3rd week: 15.07.13 – 19.07.13

Chemical-based transformation. BioBricks: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2020, E2030. Failure: E2020. Succeed: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2030. Isolate plasmid DNA by alkaline lysis. Quality cheked with spectrophotomeric analysis with NanoDrop.

4th week: 22.07.13 – 26.07.13

Transformation with isolated pladmids and BioBricks: I74609 and K864100. Isolate plasmid DNA (from I73609 and K864100) with mini-prep kit. Digest and ligation of construct: J23100 and B0034 on plasmid pSB1C3.

5th week: 29.07.13 – 1.08.13

Preparing construct: J23100 + B0034 + K864100 on plasmid pSB1A3. Measure glowing fluorescent proteins: sfGTP, sfYFP, sfCFP, sfBFP and SYFP2. Superfolded (sf) forms are made by Warsaw Team. SYFP2 is from parts registry.

6th week: 5.08.13 – 9.08.13=

Measure glowing fluorescent proteins. Banking strains with our fluorescent proteins.

7th week: 12.08.13 – 14.08.13

Transformation BioBrick: E2050. PCR: sfBFP N-terminal, sfYFP N-terminal and mCherry (J06504) C-terminal (succeed). Isolate plasmid DNA with: sfGFP (I73609), mOrange (E2050), sfCFP, sfYFP and sfBFP.

8th week: 19.08.13 – 23.08.13

PCR: sfGFP N-terminal (failure), sfCFP N-terminal (succeed) and mCherry N-terminal (succeed). Gel-out: sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal. Cloning sfCFP N-terminal, mCherry N-terminal, sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal to pSB1C3 plasmid and inserting it to bacteria. Preparing construct: J23100+B0034+E0040 – failure.

9th week: 26.08.13 – 30.08.13

Verification cloning.

Results. Marker - GeneRuler 100 bp DNA Ladder, ready-to-use (Thermo); sfYFP N 1,2,3,R1,R2,R3 - random clones of sfYFP N-terminal; sfCFP N 1,2,3,R1,R2,R3 - random clones of sfCFP N-terminal; mCherry N 1,2,3,R1,R2,R3 - random clones of mCherry N-terminal; mCherry C 1,2,3,R1,R2,R3 - random clones of mCherry C-terminal; sfBFP N 1,2,3,R1,R2,R3 - random clones of sfBFP N-terminal. Underlined - correct clones.

And great success!

PCR: sfGFP N-terminal and GFP C-terminal (various attenuation template DNA) and sfC-terminal (to versions: m6 and m12 to all of superfolded fluorescent proteins). Cloning and inserting it to bacteria. Once again preparing construct: J23100+B0034+E0040.

10th week: 2.09.13 – 7.09.13

Welcome school! Verification cloning. Unfortunately epic fail. PCR: sfGFP N-terminal, GFP N-terminal and GFP C-terminal. At least success!