Team:UNIPV-Pavia/Notebook/Week3Jul

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(New page: {{UNIPV-Pavia/Month}} __NOTOC__ <div> = Week from July 6th, to July 12nd, 2009 = <html> <table width="100%"> <tr> <td align="left" width="50%"> <a href="https://2009.igem.org/Team:UNIPV-P...)
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= Week from July 6th, to July 12nd, 2009 =
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= Week from July 13rd, to July 19th, 2009 =
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul">Next Week</a>
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== <html><font class="dayw_style">July, 6th</font></html> ==
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== <html><font class="dayw_style">July, 13rd</font></html> ==
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*We planned to dedicate this week to the assembly of an IPTG/lactose->PoPS device in order to have a lactose sensor for our project, but also a useful inducible system for general purpose applications.
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*We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:
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{|cellpadding="20"
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|R0011(E-X)
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|BOL1(E-S)
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*Gel run/cut and band purification.
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*The purified DNA gave a good result at Nanodrop, but we decided to perform the entire work on BOL1 again because we were not sure that we had extracted a pure band.
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*We stored R0011(E-X) DNA at -20°C.
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*We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).
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''Preparation of experiment with Tecan F200''
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*We picked a colony of B0030 plate (stored at +4°C) and infected 5 ml of LB + Amp.
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*We also infected 5 ml of LB + Amp with 10 ul of A1 and J23100 glycerol stocks.
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*We incubated the inocula overnight at 37°C, 220 rpm.
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== <html><font class="dayw_style">July, 7th</font></html> ==
 
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*Miniprep for BOL1 (X2).
 
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*Digestion for BOL1(E-S)(X2).
 
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*Gel run/cut/purification: none of the two BOL1 samples gave a good results in DNA quantification...we decided to cut the miniprepped plasmids again, but this time through an overnight digestion (both plasmids E-S).
 
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*In case of failure, we also infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture at 37°C, 220 rpm.
 
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*We prepared 0.5 l of LB + Amp.
 
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*We received sequencing results for:
 
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**T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".
 
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**A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.
 
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''Preparation of experiment with Tecan F200''
 
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*We diluted 1:100 the overnight cultures of A1, B0030 and J23100.
 
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*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).
 
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''Experiment with Tecan F200''
 
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* <html><u>Description</u></html>
 
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* <html><u>Purpose:</u></html>
 
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* <html><u>Materials & Methods</u></html>
 
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* <html><u>Protocol</u></html>
 
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* <html><u>Results</u></html>
 
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''Preparation of tomorrow's experiment with Tecan F200''
 
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*We picked a colony from B0030 native plate (stored ad +4°C) and infected 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.
 
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== <html><font class="dayw_style">July, 14th</font></html> ==
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== <html><font class="dayw_style">July, 8th</font></html> ==
 
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*Gel run/cut/band purification for BOL1(E-S) overnight digestion. Results: gel showed that the enzymes had cut very well and this time also the purification gave a good quantification at Nanodrop.
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== <html><font class="dayw_style">July, 15th</font></html> ==
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*We took R0011(E-X) stored ad -20°C and we were ready to ligate;)
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*Ligation:
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**A11: BOL1(E-S) + R0011(E-X) in pSB1A2
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*We incubated the ligation overnight at 16°C.
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''Preparation of experiment with Tecan F200''
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*We diluted 1:100 the overnight culture of B0030.
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''Experiment with Tecan F200 (continues the next day)''
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* <html><u>Description</u></html>
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* <html><u>Purpose:</u></html>
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* <html><u>Materials & Methods</u></html>
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* <html><u>Protocol</u></html>
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* <html><u>Results</u></html>
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== <html><font class="dayw_style">July, 9th</font></html> ==
 
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*We resuspended F2620 BioBrick from iGEM 2009 plates.
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== <html><font class="dayw_style">July, 16th</font></html> ==
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*We transformed about 40 pg of the overnight ligation and 1 ul of the resuspended F2620 BioBrick. We plated transformed bacteria and incubated the two plates overnight at 37°C.
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''End of experiment with Tecan F200 (last measurement)''
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== <html><font class="dayw_style">July, 10th</font></html> ==
 
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*A11 and F2620 overnight plates showed colonies!
 
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*Colony PCR for 6 colonies of A11 plate: the picked colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for PCR results.
 
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*We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.
 
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{|align="center"
 
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|[[Image:pv_colonypcr_A11.jpg|thumb|300px|left|Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.]]
 
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*Gel results: all the picked colonies showed the expected amplicon of a correctly ligated plasmid! We decided to prepare a glycerol stock for:
 
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**A11-1
 
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**A11-3
 
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**A11-6
 
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*Next week, only A11-1 will be used, but the other colonies will be used in case of failure.
 
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*We also prepared a glycerol stock for F2620 culture.
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== <html><font class="dayw_style">July, 17th</font></html> ==
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Revision as of 09:03, 16 July 2009

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