Team:UNIPV-Pavia/Calendar/July/settimana1
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<html><p align="center"><font size="4"><b>JULY: WEEK 1</b></font></p></html><hr><br> | <html><p align="center"><font size="4"><b>JULY: WEEK 1</b></font></p></html><hr><br> | ||
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==June, 28th== | ==June, 28th== | ||
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||MG1655 || ''E. coli'' wild type strain||LB, 37°C | ||MG1655 || ''E. coli'' wild type strain||LB, 37°C | ||
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+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==June, 29th== | ==June, 29th== | ||
- | All cultures were grown. From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a | + | All cultures were grown. From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a preliminary TECAN test. |
Cultures were MiniPrepped with the following quantifications (NanoDrop): | Cultures were MiniPrepped with the following quantifications (NanoDrop): | ||
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- | Digestion was performed at 37°C for 3 hours. Digestions were gel run. For I9 and I10 clones all | + | Digestion was performed at 37°C for 3 hours. Digestions were gel run. For I9 and I10 clones all colonies were correct, while for I7 and I8 extra bands could be observed. Since preliminar results of TECAN test didn't provide encouraging results, we decided to select other 3 colonies both for I7 and I8 and to repeat the screening, while I9 and I10 were correct at this screening, so we chose I9-1 and I10-1 as definitive I9 and I10 parts. I9 and I10 will be further screened next week. |
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- | PhaP-1 and PhaP-2 were prepared or sequencing: 13,8 ul (2x quantity) of PhaP-1 and 14,7ul (1x quantity) | + | PhaP-1 and PhaP-2 were prepared or sequencing: 13,8 ul (2x quantity) of PhaP-1 and 14,7ul (1x quantity) of PhaP-2 were dryed at 65°C and sent to BMR genomics for sequencing service. |
- | All strains received from Yale Univesity were on blotting paper disks, that were placed on an LB agar plates (with/without antiobiotic, see growth conditions), resuspended | + | All strains received from Yale Univesity were on blotting paper disks, that were placed on an LB agar plates (with/without antiobiotic, see growth conditions), resuspended with 80ul LB and then streaked. After plate streaking, disks were transferred in falcon tubes containing liquid LB. |
* '''BT340''' was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml). | * '''BT340''' was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml). | ||
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* '''BW25142''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB. | * '''BW25142''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB. | ||
* '''BW25141''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB. | * '''BW25141''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB. | ||
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+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==June, 30th== | ==June, 30th== | ||
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Inoculum of cultures for a TECAN test. From glycerol stocks: I7-1, I7-2, I8-1, I8-2 (all tese 4 colonies were not correct! we want to see if any fluorescence is observed in order to understamd if there is any unexpected GFP production), I9-1, I9-2, I10-1, I10-2. For these cultures 8ul of glycerol stock were inoculated in 2 ml Lb+Amp. | Inoculum of cultures for a TECAN test. From glycerol stocks: I7-1, I7-2, I8-1, I8-2 (all tese 4 colonies were not correct! we want to see if any fluorescence is observed in order to understamd if there is any unexpected GFP production), I9-1, I9-2, I10-1, I10-2. For these cultures 8ul of glycerol stock were inoculated in 2 ml Lb+Amp. | ||
- | Other three colonies were | + | Other three colonies were picked from I7 and I8 plates. |
{| border='1' align='center' | {| border='1' align='center' | ||
|| I7-3 || 5 ml LB+ Amp, grown ON 37°C 220 rpm | || I7-3 || 5 ml LB+ Amp, grown ON 37°C 220 rpm | ||
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All cultures were incubated ON, 37°C 220 rpm. | All cultures were incubated ON, 37°C 220 rpm. | ||
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+ | ==July, 1st== | ||
All cultures incubated yesterday night were diluted 1:100 (10ul in 1ml fresh LB+Amp) and let grown for further 3 hours at 37°C 220 rpm. Also I7-3,4 and 5 and I8-3, 4 and 5 were prepared for TECAN test (10ul were diluted in 1ml LB+Amp). After dilution, static OD was measured for these cultures and a proper dilution was perfeormed, in order to start from the desired OD of 0.02, according to the formula: | All cultures incubated yesterday night were diluted 1:100 (10ul in 1ml fresh LB+Amp) and let grown for further 3 hours at 37°C 220 rpm. Also I7-3,4 and 5 and I8-3, 4 and 5 were prepared for TECAN test (10ul were diluted in 1ml LB+Amp). After dilution, static OD was measured for these cultures and a proper dilution was perfeormed, in order to start from the desired OD of 0.02, according to the formula: | ||
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+ | <br/> | ||
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+ | <img src="https://static.igem.org/mediawiki/2010/3/35/Unipv_formula_diluizione_OD.jpg" alt="Dilution formula" title="Dilution formula"/> | ||
+ | </div> | ||
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After dilution, 3 aliquotes each of 200ul were transferred in a 96-wells plate and incubated in TECAN multi-well plate reader. Cultures were incubated at 37°C and a kinetic cycle was performed: | After dilution, 3 aliquotes each of 200ul were transferred in a 96-wells plate and incubated in TECAN multi-well plate reader. Cultures were incubated at 37°C and a kinetic cycle was performed: | ||
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Plates were incubated at 37°C ON (except BBa_J72008 that was grown at 30°C). | Plates were incubated at 37°C ON (except BBa_J72008 that was grown at 30°C). | ||
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+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==July, 2nd== | ==July, 2nd== | ||
TECAN test provided encouraging results, showing that I7-3, I7-5, I8-4 and I8-5 produced GFP. Also I9-1, I9-2, I10-1 and I10-2 confirmed the preliminar results of the precious test, in fact GFP production was observe. At a preliminary analysis, it seems that GFP production is correlated to the srength of the promoter regulating luxI production!! :) | TECAN test provided encouraging results, showing that I7-3, I7-5, I8-4 and I8-5 produced GFP. Also I9-1, I9-2, I10-1 and I10-2 confirmed the preliminar results of the precious test, in fact GFP production was observe. At a preliminary analysis, it seems that GFP production is correlated to the srength of the promoter regulating luxI production!! :) | ||
- | Other culture (I7-1, I7-2, I7-4, I8-1, I8-2, I8-3) did not produce GFP, | + | Other culture (I7-1, I7-2, I7-4, I8-1, I8-2, I8-3) did not produce GFP, so they were thrown away. |
A furhter screening were performed on I7-3, I7-5, I8-4, I8-5, I9-1 and I10-1. | A furhter screening were performed on I7-3, I7-5, I8-4, I8-5, I9-1 and I10-1. | ||
- | For this reason, | + | For this reason, MiniPrep was performed for the following colonies, with the following quantiications: |
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Inoculum of <partinfo>BBa_J72008</partinfo> from LB agar plate (one colony in 3ml LB+Amp 50), grown at 30°C 220 rpm overnight. | Inoculum of <partinfo>BBa_J72008</partinfo> from LB agar plate (one colony in 3ml LB+Amp 50), grown at 30°C 220 rpm overnight. | ||
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+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==July, 3rd== | ==July, 3rd== | ||
Glycerol stock for <partinfo>BBa_J72008</partinfo> was prepared. | Glycerol stock for <partinfo>BBa_J72008</partinfo> was prepared. | ||
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+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
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<!-- table previous next week --> | <!-- table previous next week --> | ||
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Latest revision as of 16:52, 24 October 2010
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