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Revision as of 11:53, 11 September 2017
10/07/2017
- Extracted interlab devices from kit and made up spread plates.
11/07/2017
- Streak plates of DH5α and MG1655.
- Overnight cultures for interlab devices- 5ml + chloramphenicol + one colony.
12/07/2017
- Overnight cultures for wild type (DH5α, MG1655) - 5ml LB + one colony.
- Glycerol stocks (DH5α, MG1655).
- Miniprep of interlab devices (and pBAD).
- Qubit of devices (and pBAD).
13/07/2017
- Glycerol stocks of wild types DH5α and MG1655.
- Made 5x 200ml of LB (autoclaved)
- Genomic DNA prep of DH5α and MG1655.
- Qubit for wild types.
14/07/2017
- Introduction to TECAN plate reader.
- Competent cell preparation for DH5α (see protocol).
- Measurements for interlab.
17/07/2017
- Interlab device transformations (expt. 3)
- Restriction digest for insertion of fim sections into plasmids (Expts. 1+2)
- Stored fim plasmids.
18/07/2017
- Liquid overnights for interlab (error expt.6)
- PCR and electrophoresis (Taq polymerase) of fim genes (D,H and A) - confirms genes in DH5α (bad news) (expt.4)
19/07/2017
- Transformed plasmids from expt.2 into cells (expt.5)
- Liquid overnights for interlab.(expt.9)
- Electrophoresis of fim operon from expt.4 (expt.7)
- PCR (Phusion) of whole operon. (expt.8)
20/07/2017
- Interlab measurements.(expt.10)
- Miniprep and Qubit for fim constituents. (expt.11)
- Competent cell prep (expt.12)
- Genomic DNA extraction of Top10 strain (expt. 13)
- Electrophoresis for PCR from expt.8 (expt.14)
21/07/2017
- Prepared DNA from miniprep for sequencing (to confirm that transformation worked)
- Competent cell prep (expt.12)
- Genomic DNA extraction of Top10 strain (expt. 13)
- Electrophoresis for PCR from expt.8 (expt.14)
24/07/2017
- Interlab data analysis
- Overnights for Top10
25/07/2017
- Genomic extraction from Top10 (x2)
- DNA extraction from gel band (MG1655 operon)
- PCR of Top10 and MG1655 fim genes.
26/07/2017
- Electrophoresis of Top10 and MG1655
- One pot cloning of fimAIC, D, and FG with promoter.
- Transformed fim containing plasmids into DH5ɑ
- Streaked MG1655 and Top10 for Bioimaging.
27/07/2017
- Overnights for MG1655 and Top10 for imaging.
- Transformed MoClo products into DH5ɑ.
28/07/2017
- Negative stain and transmission electron microscopy of MG1655 and Top10 samples.
31/07/2017
- Overnights of MG1655, Top10 and MoClo products (x8) from restriction digest of fimH constructs and ligation into plasmids.
01/08/2017
- Transformed fim H constructs (x10) into dh5alpha
- One pot: prepared for sequencing, made glycerol stocks, miniprep and qbit.
- Dh5alpha overnights, made competent cells of top10.
02/08/2017
- Restriction digest of operon MoClo products (for gel)
- Prep. for fim operon sequencing
03/08/2017
- MoClo of fimH constructs.
04/08/2017
- Sequencing results confirm successful plasmid construction.
- Transformation of MoClo products into dh5alpha.
07/08/2017
- Transformation of fimH MoClos and preparation of fim operon for sequencing.
08/08/2017
- Top 10 competent cells made
10/08/2017
- Miniprep and Qubit of pRha+fimH+psB1A3 constructs.
- Overnights cultures of pAra+fimop(pX3’6’0’0), J23100+fimop(pX3’6’0’0), J23100+fimop(PSB1A3)
11/08/2017
- Prep of pRha+fimH miniprep DNA for sequencing.
- Qubit of overnight cultures: (J23100 x 2 + pAra) + fimH.
14/08/2017
- Restriction digest of Fim operon in: pX3’6’0’0 with J23100 promoter, pX3’6’0’0 with pAra, pSB1A3 with J23100.
- The products of the restriction digest were ran on the electrophoresis and only the pSB1A3 product was successful. Only two pSB1A3 plasmids were sent for sequencing (5+6).
- The pRha+fimH+psB1A3 constructs were also sent for sequencing (13 constructs).
15/08/2017
- Poured CAM plates, Restriction digest of pAra_fimoperon_pSB1A3, J23100_fimoperon_ pSB1A3 and the pX3’6’0’0 plasmid, followed by a gel electrophoresis and gel extraction of the DNA (the wrong gel bands were extracted, so Chloe did it again for us).
- Transformation of the FimH constructs in the pSB1C3 and pSB1A3 into DH5a.
16/08/2017
- Gel extraction of the bands (after that we found out the bands we extracted were the wrong ones).
- Overnights of FimH constructs in the pSB1C3 and pSB1A3 into DH5a.
17/08/2017
- Mini prep and Qubit from overnights of FimH constructs and transformation of the ligation from the gel extraction on CAM plates that Chloe did.
21/08/2017
- Miniprep, glycerol stocks and qubit of J23100 and pAra fim_op.
- Diagnostic digest and electrophoresis of above - shows successful construction and transformation of plasmids.
22/08/2017
- Double transformations of two plasmids into Top10.
- Transformation of fimH constructs into pEX1A3.
- Transformation for UV experiments.
23/08/2017
- Liquid overnights for previous transformations.
24/08/2017
- First attempt at induction of fimH constructs.
28/08/2017
- Overnights of transformed top10, WT top10 and WT MG1655
29/08/2017
- Revised induction protocol
- Measured construct fluorescence in TECAN
30/08/2017
- Sand protocol
- First attempt at agglutination protocol
31/08/2017
- Induction protocol
1/08/2017
- Samples run through the TECAN
4/08/2017
- Overnight cultures of constructs
5/08/2017
- Induction protocol
- Agglutination protocol
- Successful transformation of mouse metallothionein
6/09/2017
- Samples run through FACS and fluorescent cells sorted
- Sorted cells plated on agar in 7x8 grid
7/09/2017
- Induction protocol
8/09/2017
- Dialysis trial run
11/09/2017
- Induction protocol
