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Team:Cambridge/Diary/Week 13
From 2012.igem.org
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Contents |
Monday
- Ratiometrica: grow O/N cultures for rough IPTG induction tomorrow, ordered primers to convert into biobrick
Tuesday
- Ran PCR for making SPOBB biobrick, gel yielded insufficient results. Redesigned and reordered primers
- Ratiometrica: rough IPTG induction with 8 different concentrations of IPTG, after 3 hours of incubation there was unfortunately no obvious results (i.e. no visible difference between different concentrations/induced and uninduced smaples), will leave them growing overnight and observe again tomorrow; sequencing primers arrived so the plasmid is sent off to sequencing, will have results tomorrow morning; more O/N cultures grown for miniprep to put into Bacillus tomorrow.
Wednesday
- Ratiometrica: Sequencing results came back- good news: CFP is inserted correctly after pSPAC, bad news: more uncertainties as to whether the K143053 and YFP are inserted correctly. Since the cells shows clear YFP under the fluorescent microscope, we cannot really tell what is wrong at the moment, more sequencing primers are ordered to verify the sequence further along the construct. Tom ran a restriction digest with the new miniprep products and they showed exactly the expected results- so we are now more certain that we have got the right construct! IPTG induction in liquid cultures yesterday really didn't give reliable results, so we have now plated cells on IPTG plates for induction and hopefully we will see results tomorrow. Bacillus has also been transformed- it's been a while since we've done Bacillus transformation, and we will know if it works tomorrow. Primers arrived today morning and PCR has been done to convert ratiometrica into a biobrick! We are having some slight problems with the linearised backbone but are slowly working our way around it... hopefully it will all come into place.
Thursday
- Ratiometrica: Yesterday's IPTG induction on plates seemed to have worked! Cells on IPTG plate showed much more CFP than those on the uninduced plates. We are growing O/N cultures in minimal medium so we can test them on a plate reader to get more accurate results and to characterise the part... We are still having trouble with the linearised backbone, plan A is that this currently running PCR cycle will work out and we will have the suitable backbone, plan B is to wait for the correct primers to hopefully come in asap! Bacillus transformation from yesterday produced 1 bacillus colony- it is hard to tell if it is the thing we are looking for as ECFP and EYFP signals are rather poor in Bacillus without the ComGA codons, but there is definitely a little bit of CFP and YFP. We have grown O/N culture of that in LB and will probably extract the genomic DNA then PCR as a verification test. Time is running out...
Friday
Saturday
Sunday
