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Team:UNIPV-Pavia/Calendar/September/settimana1
From 2010.igem.org
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(→August, 31st) |
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==August, 31st== | ==August, 31st== | ||
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| + | Glycerol stocks were prepared for | ||
| + | *I29-1 | ||
| + | *I29-2 | ||
| + | *I29-3 | ||
| + | picked yesterday from I29 ligation agar plate. | ||
| + | Remaining culture was MiniPrepped to purify DNA. | ||
| + | |||
| + | {| | ||
| + | |'''Culture''' || '''Quantification''' | ||
| + | |- | ||
| + | | I29-1 || 149,6 ng/ul | ||
| + | |- | ||
| + | | I29-2 || 110,3 ng/ul | ||
| + | |- | ||
| + | | I29-3 || 91,0 ng/ul | ||
| + | |} | ||
| + | |||
| + | DNA was digested for 3 hours at 37°C as follows: | ||
| + | {| border="1" align='center' | ||
| + | | ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' | ||
| + | |- | ||
| + | | I29-1 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5 | ||
| + | |- | ||
| + | | I29-2 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5 | ||
| + | |- | ||
| + | | I29-3 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5 | ||
| + | |} | ||
| + | and than gel run to check the length of ligations. | ||
| + | ---- | ||
| + | Sudan Black staining protocol for BioPlastic: | ||
| + | |||
| + | Sudan Black and Safranine stain were prepared: | ||
| + | *Sudan Black 0,3% in EtOH 70% (0,1 g Sudan Black in 33,3 ml EtOH 70%) | ||
| + | *Safranine 0,5% in H20 (1,25 g Safranine in 250 ml ddH20) | ||
| + | |||
| + | Cultures of PBHR68 and RBS were diluted 1:100 this morning and let grown till they reached an OD600 0,06. When they reached this optical density, they were induced with 1mM IPTG and following samples were prepared: | ||
| + | |||
| + | *RBS (C-) | ||
| + | *PBHR68 with NOTHING added | ||
| + | *PBHR68 with 2% glycerol added | ||
| + | *PBHR68 with 1mM IPTG added | ||
| + | *PBHR68 with 2% glycerol and 1mM IPTG added | ||
| + | |||
| + | After 8 hours, microscope slides were prepared with the usual protocol. | ||
| + | |||
| + | ---- | ||
| + | |||
| + | |||
| + | |||
| + | |||
PCR from colony for I47, I48, I49 ligations. Three colonies were picked from each plate, and they were also let grow in LB+Amp to make glycerol stocks of the positive ones. | PCR from colony for I47, I48, I49 ligations. Three colonies were picked from each plate, and they were also let grow in LB+Amp to make glycerol stocks of the positive ones. | ||
| - | [[Image: | + | |
| + | |||
| + | |||
| + | |||
| + | [[Image:UNIPV10_31_8_10_ColonyPCR_I47_I48_I49_and_I29_screening.jpg|thumb|200px|center|Colony PCR for I47, I48, I49 ligations and screening for I29-1, I19-2 ans I29-3.]] | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
Revision as of 10:57, 31 August 2010
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