Notebook
Day 1
- Binding assay of gtfC to dynabeads
- Inhibition study of D3 PCR
- Dephosphorylation of gtfC2 + psB1C3
- Gel electrophoreis, gel extraction, ligation
Day 1
- Miniprep of gtfC part 2
- Ligation of gtfC part 2 + gtfC part 1
- Ligation of gtfC part 1 + gtfC part 2
Day 1
- Checked sequencing results
- Checked buffer's composition
Day 2
- Restriction analysis of gtfC 2 + 1
- Enzyme assay with Miller reagents
- Picked 4 more colonies of Spap1 and 1 pick gtfC2+1
Day 3
- Miniprep
- Restriction digest, gelelectrophoresis
Day 4
- Restriction analysis of gtfC 2+1 with EcoRI, HindIII, PstI
- Restriction analysis of gtfC 2+1 with EcoRI, HindIII
- Sent in Pick 8 of SpaP 1+2
- Sent in Pick 1 of gtfC 1+2
Day 1
- Restriction digest
- Gel extraction
- Ligation over night of gtfC part 2 in psB1C3
- PCR of amplified gtfC part 1
Day 2
- D3 library amplification
- D3 library inhibition assay
- Poured new agar plates
- PCR amplification
Day 3
Day 1
- Restriction
- Gel extraction
- Over night ligation of gtfc2 in psB1C3 and PCR amplified gtfC1
Day 2
- Transformation of NEB with ligates
- Miller assay
- Beads coupling assay
Day 3
- Prepared cultures for miniprep
- Checked if protein inhibits the D3 library’s PCR
Day 4
- Miniprep
- Restriction analysis
- Sent in DNA
- PCR for Spap 1 and 2
Day 1
- PCR for amplification of the parts
- Changed the buffer of enzyme with the Mondelli buffer
Day 2
- Miniprep of gtfC part 2
- Ligation of gtfC part 2 + gtfC part 1
- Ligation of gtfC part 1 + gtfC part 2
Day 3
Day 4
- Performing restriction of each part with EcoRI and PstI
- Restriction of psB1C3 plasmid
- Restriction of each SpaP part with NheI, PCR clean up and subsequent ligation
- Restriction of each gtfC part with HindIII, PCR clean up and subsequent ligation
- Ligation on higher concentrations
Day 5
- Picking colonies
- Binding assay in Mondelli buffer without BSA and tween
- PCR of gtfC part 1
Day 1
- Adding SDS into the Miller-reagent
- Measuring the pH of the Mondelli-buffer
- Glucan assay with new concentrations
- Checking the pH of the Mondelli-buffer
Day 2
- Miller assay with Mondelli buffer
- Preculture for IPTG induction
- PCR of parts
- Binding Enzyme to Ni-NTA-beads
- Calibrating pipettes
Day 3
- IPTG Induction
- Ligations and transformations with a higher concentration in DNA
- Running a gel of d3 library
Day 4
- PCR amplfication of IDT parts with newly arrived primers
- Evaluating IPTG induction and gel of D3 library
- Testing Ni-NTA-beads’ binding capacity for our enzyme
Day 1
- Testing the Miller-reagent
- Gel electrophoresis
Day 2
- Redid restriction of pick 1, 2, 3, 4 of gtfC part 2+1
Day 3
- Miniprep of gtfC part 2
- Miniprep of amplified parts
Day 4
- IPTG Induction of BL21 promoter construct
- Enzyme assay
Day 1
- Minipreps of 5 Spap2-picks
- Dephosphorylation of gtfC2 + psB1C3
- Ligation of gtfC 1 + 2
- Restriction of:
- Picks 5 - 9 of SpaP part 2 (for Restriction-Analysis)
- GTFc I and II (only HindIII)
- validated (GTFc II + psB1C3) & GTFc II (EcoRI & HindIII)
Day 2
- Ligation and transformation of gtfC1 into vector+gtfC2
- Restriction analysis of SpaP 2
Day 3
- Picking of colonies (gtfC1+2)
- Inoculation in LB-media
Day 4
- Miniprep and restriction for later restriction analysis
Day 1
- Picking and inoculating miniprep results
- Restriction analysis
- Inoculating LB medium with validated gtfC part 2 bacteria
Day 2
- Miniprep of gtfC part 2
- Ligation of gtfC part 2 + gtfC part 1
- Ligation of gtfC part 1 + gtfC part 2
Day 1
- Ligation of gtfC part 2 into psB1C3
- Ligation of gtfC part 1+2 int psB1C3
Day 2
- Transformation in NEB Turbo (part 2)
- Transformation in NEB Turbo and BL21 (part 1+2)
Day 3
- Picking clones (4-5 each plate, 10 from part 1+2) in TB+Kanamycin
- Miniprep of cultures
Day 4
- Restriction analysis of the results from miniprep
- Sending in the results for sequencing
Day 1
- Restriction analysis of SpaP2 and gtfC2
Day 2
- Ligation ans transformation of IDT genes (part1 of SpaP)
Day 3
- Miniprep of five picks of SpaP fragment and SpaP part 2
- Miniprep of the other colonies
- Transformation of BL21 with pick 4 of promoter B1C3 construct
Day 4
- Transformation of promoter B1C3 construct into BL21
- Ligation and transformation of SpaP part 2 and 1 into NEB Turbo bacteria
Day 5
- Preparation of Glycerol stocks
- Restriction digest
- Sent DNA for sequencing
Day 1
- Cutting, ligating and transformation of IDT genes (fragment of SpaP)
- Added canamycin to the agar LB and poured plates
Day 2
- Miniprep of three picks of SpaP fragment
- Cutting, ligating and transformation of SpaP part2 and gtfC
Day 3
- Miniprep of three picks of each SpaP and gtfC
- Restriction analysis of SpaP fragment's picks and sending to sequencing
- Competent BL21 bacteria prepared
Day 4
- Restriction analysis of part 2 of SpaP and gtfC
- Results sent to sequencing
Day 1
- Preparing LB medium and CaCl₂ buffer
- Making new plates
- Starting pre-culture of BL21 bacteria
Day 2
- Preparing competent bacteria
Day 3
- Testing the competence of the bacteria
- Preparing agar plates
Day 1
- Starting a pre-culture with BL21 bacteria in non-validate box
Day 2
- Production of competent bacteria
- Transformation of BL21 bacteria with the promoter construct and with the standard C1B3-Plac-mRFP1 as a positive control
Day 3
- Picking colonies of our construct and the positive control into TB medium
Day 4
Day 5
- Picking colonies and inoculating
- Making glycerol stocks
- Gel electrophoresis
- Sending DNA for sequencing
Day 1
- Transformation of NEB Turbo bacteria
Day 2
- Picking colonies and inoculating
- Making glycerol stocks
- Gel electrophoresis
- Sending DNA for sequencing
Day 3
- Planning IPTG induction
- Starting a pre-culture
Day 4
- IPTG induction at OD600 ~ 0.4
Day 5
- Picking colonies and inoculating
- Making glycerol stocks
- Gelelectrophoresis
- Sending DNA for sequencing
Day 1
Day 2
- Digest
- DNA extraction
- Dephosphorylation of the backbone
- Ligation
- Making INOUE buffer for new competent bacteria
- Making SOB
Day 1
- Sequencing sample 1.2
- Transformation with and without IPTG
Day 2
- Checking TB transformation
Day 3
Day 1
- Inoculation of the promoter construct
- Miniprep
Day 2
Day 3
- Re-Measurement of C3-IDT-RFP
- Restriction analysis
- Inoculation of plate 1 and 2
Day 4
- Making glycerol stocks
- Miniprep
- Restriction analysis
